Protein having antithrombotic activity and method for producing the same

ABSTRACT

A method for producing a protein having an antithrombotic activity, which comprises replacing, in a protein that has an amino acid sequence having a homology of not less than 30% to the amino acid sequence of SEQ ID NO: 1 and forms a higher order structure composed of a first β strand (β1), a first α helix (α1), a second α helix (α2), a second β strand (β2), a loop, a third β strand (β3), a fourth β strand (β4) and a fifth β strand (β5) in this order from the amino terminus, at least one amino acid residue in a region from α2 to β2 and/or a region from β3 to β4 so that electric charge of the amino acid residue is changed towards positive direction.

This patent application claims foreign priority benefits. Specifically,this patent application claims the benefit of the filing date under 35U.S.C. 119 of Japanese Application No. 2000-305279, filed Oct. 4, 2000.

BACKGROUND OF THE INVENTION

The present invention relates to a protein having an antithromboticactivity and a method for producing the same. The present invention alsorelates to DNA coding for the protein and a drug containing the proteinas an active ingredient.

The number of patients with thromboses such as myocardial infarction andcerebral thrombosis, in particular, arterial thrombosis, is high in theworld, and these are very important diseases to be treated. In an earlystage of onset of arterial thrombosis, von Willebrand factor in bloodbinds to subendothelial tissues (collagen etc.) exposed due toimpairment of vascular endothelial cells, and a membrane glycoprotein onplatelets, glycoprotein Ib, binds to the von Willebrand factor. Thus,the platelets adhere to blood vessel walls and they are activated (J. P.Cean et al., J. Lab. Clin. Med., 87, pp.586-596, 1976; K. J. Clemetsonet al., Thromb. Haemost., 78, pp.266-270, 1997). Therefore, it is animportant target for antithrombotic drugs for treating or preventingthromboses to inhibit the binding of von Willebrand factor andglycoprotein Ib. However, there are few substances that have proven toexhibit antithrombotic property by inhibiting the binding of theseproteins, and such drugs have not been used in clinical practice. It hasbeen reported that a recombinant protein VCL that has a sequence of the504th to 728th amino acid residues of the amino acid sequence of vonWillebrand factor shows an antithrombotic action by inhibiting thebinding of von Willebrand factor and glycoprotein Ib (K. Azzam et al.,Thromb. Haemost., 73, pp.318-323, 1995). Further, it has also beenreported that a monoclonal antibody AJvW-2 directed to human vonWillebrand factor exhibits an antithrombotic activity by specificallybinding to von Willebrand factor without showing hemorrhagic tendency(S. Kageyama et al., Br. J. Pharmacol., 122, pp.165-171, 1997). It hasalso been shown that a monoclonal antibody 6B4 directed to glycoproteinIb has an antithrombotic action in animal models (N. Cauwenberghs etal., Atherioscler. Thromb. Vasc. Biol., 20, pp.1347-1353, 2000).Furthermore, the protein AS1051 originating from snake venomspecifically binds to platelet glycoprotein Ib to similarly exhibitantithrombotic property without showing hemorrhagic tendency (N. Fukuchiet al., WO95/08573).

Meanwhile, the binding of von Willebrand factor and glycoprotein Ib isnot observed under a usual condition, but is considered to occur onlyunder a condition where shear stress is induced, such as a condition ina blood flow (T. T. Vincent et al., Blood, 65, pp.823-831, 1985).However, as a method for artificially observing the binding of theseproteins, there are known addition of an antibiotic, ristocetin (M. A.Howard and B. G. Firkin, Thromb. Haemost., 26, pp.362-369, 1971) andaddition of a protein originating from snake venom, botrocetin (M. S.Read et al., Proc. Natl. Acad. Sci. USA., 75, pp.4514-4518, 1978). Thatis, both of the substances are considered to cause a structural changeof von Willebrand factor by binding to a specific site of von Willebrandfactor, thereby causing the binding of von Willebrand factor andglycoprotein Ib, which does not occur under a usual condition.

As proteins originating from snake venom, there are known, in additionto the aforementioned AS1051 (derivative of the α-chain of a proteinoriginating from Crotalus horridus horridus snake venom, CHH-B) and itsoriginal protein, CHH-B, many glycoprotein Ib-binding proteins such asalboaggregin, echicetin, mamushigin, jararaca-GPIbBP and proteinsoriginating from Cerastes cerastes. Many of these proteins have aheterodimeric structure, and the amino acid sequences of their subunitsshow a homology of not less than 30%. Furthermore, they are proteins inwhich all of subunits show an amino acid sequence homology of not lessthan 30% to the CHH-B α-chain (R. K. Andrews et al., Biochemistry, 35,pp.12629-12639, 1996; Y. Fujimura et al., Thromb. Haemost., 76,pp.633-639, 1996).

While such glycoprotein Ib-binding proteins originating from snake venomthat inhibit the binding of glycoprotein Ib and von Willebrand factorand monoclonal antibodies directed to von Willebrand factor orglycoprotein Ib are known to exhibit an antithrombotic action asdescribed above, some of proteins originating from snake venom that bindto a platelet membrane glycoprotein, glycoprotein IIb/IIIa, disintegrins(T. Matsui et al., Biochem. Biophys. Acta, 1477, pp.146-56, 2000) andmonoclonal antibodies directed to glycoprotein IIb/IIIa (A. M. Lincoffet al., J. Am. Coll. Cardiol., 35, pp.1103-1115, 2000) have also beenshown to exhibit an antithrombotic activity in animal experiments orclinical practice. For example, a peptide prepared from a disintegrinsequence, Eptifibatide (integrilin), has been shown to have clinicalefficacy as an antithrombotic drug. Further, a chimerized monoclonalantibody directed to glycoprotein IIb/IIIa, Abciximab (ReoPro), is alsowidely used as an antithrombotic drug in clinical practice and itsstrong antithrombotic action and its therapeutic action for acutecoronary syndromes have been reported (M. Madan et al., Circulation, 98,pp.2629-2635, 1998).

In addition to the above proteinaceous substances, low molecular weightorganic compounds that bind to a platelet membrane glycoproteins andinhibit their function are known with respect to glycoprotein Ib (N.Fukuchi et al., WO99/54360; W. Mederski et al., WO00/32577; H. Matsunoet al., Circulation, 96, pp.1299-1304, 1997) and glycoprotein IIb/IIIa(E. J. Topol et al., Lancet, 353, pp.227-231, 1999). Among these, someof glycoprotein IIb/IIIa antagonists are clinically used, but they havenot been shown to have efficacy as high as that of Abciximab (ReoPro)(E. J. Topol et al., Lancet, 353, pp.227-231, 1999; M. Madan et al.,Circulation, 98, pp.2629-2635, 1998).

As described above, proteins that bind to platelet membrane proteinsinvolved in thrombogenesis such as glycoprotein Ib and glycoproteinIIb/IIIa and inhibit their functions in thrombogenesis are useful asantithrombotic drugs, and many exogenous proteins have been developed asantithrombotic drugs. Among these, a chimerized monoclonal antibodydirected to glycoprotein IIb/IIIa, Abciximab (ReoPro), shows highclinical efficacy. However, the following some conditions are stillrequired to use proteinaceous substances, in particular, exogenousproteins as clinically usable drugs.

(1) High Binding Activity to Target

In the case of Abciximab (ReoPro), a high binding activity to platelets(glycoprotein IIb/IIIa) can be mentioned as one of the reasons for itshigh efficacy (R. M. Scarborough et al., Circulation, 100, pp.437-444,1999). That is, it is considered that the administered Abciximab(ReoPro) firmly binds to platelets and as a result, it exists in bloodtogether with platelets for a long period, thereby showing drug efficacyfor a long period.

(2) Long Half-life/High Drug Efficacy Retention in Blood

For administration of a proteinaceous drug, in particular, a drug thatis not originally an endogeneous substance existing in the organisms,repetitive administration is generally difficult and a singleadministration is usually performed. Therefore, drug efficacy must bemaintained for a certain long period and long half-life and/or high drugefficacy retention in blood is required.

(3) Low Antigenicity

Even when a single administration is performed, low antigenicity isrequired so that an excessive antigen-antibody reaction should notoccur.

(4) Useful Actions in Addition to Main Action

There have been reported that Abciximab (ReoPro) actually has a bindingaction directed to other proteins such as αvβ3 integrin and Mac-1 inaddition to an inhibitory action directed to glycoprotein IIb/IIIa (B.S. Coller, Thromb. Haemost., 82, pp.326-336, 1999). It is consideredthat this secondary action is one of the reasons for high clinicalefficacy. That is, clinical efficacy of a drug may be increased byacting on several targets other than a single target.

It has been reported that a drug for inhibiting the binding ofglycoprotein Ib and von Willebrand factor has a low risk of hemorrhagecompared with a drug for inhibiting the function of glycoproteinIIb/IIIa (S. Kageyama et al., Br. J. Pharmacol., 122, pp.165-171, 1997),and therefore it can be a useful antithrombotic drug. Among theaforementioned proteins that inhibit the binding of glycoprotein Ib andvon Willebrand factor, monoclonal antibodies generally have a highbinding activity (affinity) to a target and can satisfy the aboverequirements (2) and (3) if they are modified into a chimera antibody ora humanized antibody. On the other hand, it is considered that proteinsother than the monoclonal antibodies, for example, a glycoproteinIb-binding protein originating from snake venom have a low bindingactivity (affinity) to their targets. For example, when theanti-platelet activity disclosed for a protein derivative originatingfrom snake venom, AS1051 (N. Fukuchi et al., WO95/08573), is comparedwith that of a monoclonal antibody, AJvW-2 (S. Kageyama et al., Br. J.Pharmacol., 122, pp.165-171, 1997), the binding activity (affinity) ofAS1051 on a molar concentration basis is calculated to be about{fraction (1/10)} based on the fact that the efficacy is shown at almostthe same concentration (weight concentration), and the molecular weightof AS1051 is about 15,000 Da and that of the monoclonal antibody about150,000 Da.

Further, the present inventors found that, as shown in the examplesdescribed later, repetitive administration of AS1051 produces antibodiesfor AS1051 as an antigen and subsequent administration thereof causedplatelet decrease that was considered to be attributable to the antibodygeneration.

That is, in order to clinically use glycoprotein Ib-binding proteinsoriginating from snake venom such as AS1051 as antithrombotic drugs,they must further be improved for the aforementioned requirements (1) to(3).

SUMMARY OF THE INVENTION

The inventors of the present invention successfully elucidated a crystalstructure of a glycoprotein Ib-binding protein originating from snakevenom, AS1051, by preparing crystals of a specific mutant AS1051 andanalyzing them by X-ray diffraction analysis, and thus identified astructure unique to AS1051. Moreover, they successfully improvedglycoprotein Ib-binding proteins such as AS1051 by modifications basedon the structure. That is, they found a method for improving a proteinso that the protein should satisfy the aforementioned four kinds ofproperties, which were considered to be required to use an exogenousprotein as a clinically applicable drug, i.e., (1) a high bindingactivity to target, (2) long half-life/drug efficacy retention in blood,(3) low antigenicity and (4) useful actions, in addition to its mainaction, as well as such an improved protein. Thus, they accomplished thepresent invention.

The present invention provides a method for producing a protein havingan antithrombotic activity, which comprises replacing, in a protein thathas an amino acid sequence having a homology of not less than 30% to theamino acid sequence of SEQ ID NO: 1 and forms a higher order structurecomposed of a first β strand (β1), a first α helix (α1), a second αhelix (α2), a second β strand (β2), a loop, a third β strand (β3), afourth β strand (β4) and a fifth β strand (β5) in this order from theamino terminus, at least one amino acid residue in a region from α2 toβ2 and/or a region from β3 to β4 so that electric charge of the aminoacid residue is changed towards positive direction (hereafter alsoreferred to as the “production method of the present invention”).

In the production method of the present invention, electric charge ispreferably changed towards positive direction by replacing at least oneacidic amino acid residue in the region from α2 to β2 and/or the regionfrom β3 to β4 with a neutral amino acid residue.

In the production method of the present invention, the proteinpreferably originates from Crotalus horridus horridus.

Further, it is preferred that the region from α2 to β2 in the proteincorresponds to the sequence of the amino acid numbers 47 to 72 in theamino acid sequence of SEQ ID NO: 1 and the region from β3 to β4corresponds to the sequence of the amino acid numbers 94 to 111 in theamino acid sequence of SEQ ID NO: 1. In this embodiment, it is preferredthat at least one acidic amino acid residue of which a carbon atomexists within 10 Å from the α carbon atom of the arginine residue of theamino acid number 103 in the amino acid sequence of SEQ ID NO: 1 isreplaced with a neutral amino acid residue. Further, the acidic aminoacid residue preferably is at least one residue selected from theaspartic acid residue of the amino acid number 54, the aspartic acidresidue of the amino acid number 101 and the glutamic acid residue ofthe amino acid number 106 in the amino acid sequence of SEQ ID NO: 1.

The production method of the present invention may further comprisedeleting a region containing the loop structure existing between β2 andβ3 in such a manner that the higher order structures of β2 and β3 aremaintained, or replacing the region with one or more amino acidresidue(s) in a number required to maintain the higher order structuresof β2 and β3, said amino acid residue(s) being selected from the groupconsisting of a glycine residue, an alanine residue, a serine residueand a cysteine residue. Preferably, the region containing the loopstructure existing between β2 and β3 is replaced with an amino acidsequence composed of four glycine residues.

The production method of the present invention preferably furthercomprises bonding a polyoxyalkylpolyol group to the protein. Preferably,the protein contains a cysteine residue corresponding to a cysteineresidue of the amino acid number 81 in the amino acid sequence of SEQ IDNO: 1, and the polyoxyalkylpolyol group is bonded to this cysteineresidue. The polyoxyalkylpolyol group is preferably a polyethyleneglycol group.

The present invention also provides a protein having an antithromboticactivity, which has an amino acid sequence showing a homology of notless than 30% to the amino acid sequence of SEQ ID NO: 1 and forms ahigher order structure composed of a first β strand (β1), a first αhelix (α1), a second α helix (α2), a second β strand (β2), a loop, athird β strand (β3), a fourth β strand (β4) and a fifth β strand (β5) inthis order from the amino terminus, and wherein at least one amino acidresidue in a region from α2 to β2 and/or a region from β3 to β4 isreplaced so that electric charge of the amino acid residue in theregions is changed towards positive direction, said protein being thefollowing (a) or (b) (hereafter also referred to as the “protein of thepresent invention”):

(a) a protein, in which the region from α2 to β2 has the sequence of theamino acid numbers 47 to 72 in the amino acid sequence of SEQ ID NO: 1and the region from β3 to β4 has the sequence of the amino acid numbers94 to 111 in the amino acid sequence of SEQ ID NO: 1;

(b) the protein according to (a), in which substitution, insertion ordeletion of one or several amino acid residues is included in the regionfrom α2 to β2 having the sequence of the amino acid numbers 47 to 72 inthe amino acid sequence of SEQ ID NO: 1 and/or the region from β3 to β4having the sequence of the amino acid numbers 94 to 111 in the aminoacid sequence of SEQ ID NO: 1.

The protein of the present invention preferably comprises an amino acidsequence of the following (A) or (B):

(A) the amino acid sequence of the amino acid numbers 47 to 111 in theamino acid sequence of SEQ ID NO: 1;

(B) the amino acid sequence according to (A), in which the cysteineresidue of the amino acid number 81 in the amino acid sequence of SEQ IDNO: 1 is replaced with an alanine residue.

The protein of the present invention may have the amino acid sequence inwhich a region containing the loop structure existing between β2 and β3is deleted in such a manner that the higher order structures of β2 andβ3 are maintained, or the region is replaced with one or more amino acidresidue(s) in a number required to maintain the higher order structuresof β2 and β3, said amino acid residue(s) being selected from the groupconsisting of a glycine residue, an alanine residue, a serine residueand a cysteine residue. The region preferably has the sequence in whichthe region containing the loop structure existing between β2 and β3 isreplaced with an amino acid sequence composed of four glycine residues.

The protein of the present invention preferably has a sequence in whichat least one acidic amino acid residue of which a carbon atom existswithin 10 Å from the α carbon atom of the arginine residue of the aminoacid number 103 in the amino acid sequence of SEQ ID NO: 1 is replacedwith a neutral amino acid residue. In this embodiment, it is preferredthat the acidic amino acid residue to be replaced is composed of atleast one residue selected from the aspartic acid residue of the aminoacid number 54, the aspartic acid of the amino acid number 101 and theglutamic acid residue of the amino acid number 106 in the amino acidsequence of SEQ ID NO: 1.

Preferably, the protein of the present invention is bonded to apolyoxyalkylpolyol group. The protein of this embodiment preferablycontains a cysteine residue corresponding to the cysteine residue of theamino acid number 81 in the amino acid sequence of SEQ ID NO: 1, and thepolyoxyalkylpolyol group is bonded to this cysteine residue. Thepolyoxyalkylpolyol group is preferably a polyethylene glycol group.

The present invention also provides a DNA coding for the protein of thepresent invention (hereafter also referred to as the “DNA of the presentinvention”), as well as a method for producing the protein of thepresent invention, which comprises steps of culturing a hostmicroorganism transformed with the DNA of the present invention andcollecting a protein encoded by the DNA from a culture and a method forproducing the protein of the present invention, which comprises steps ofculturing a host microorganism transformed with the DNA of the presentinvention, collecting a protein encoded by the DNA from a culture andbonding a polyoxyalkylpolyol group to the collected protein.

The present invention further provides a drug containing the protein ofthe present invention as an active ingredient. Also, the presentinvention provides a pharmaceutical composition comprising the proteinof the present invention and a pharmaceutically acceptable carrier, anda use of the protein of the present invention for the manufacture of amedicament.

According to the present invention, a glycoprotein Ib-binding proteinoriginating from snake venom can be improved to obtain a protein having(1) higher activity, (2) higher drug efficacy retention, (3) lowerantigenicity, (4) thrombin-induced aggregation inhibitory action inaddition to its main action, i.e., an inhibitory activity for binding ofglycoprotein Ib and von Willebrand factor, and so forth, and theimproved protein can be utilized as a more effective antithromboticdrug.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 shows a three-dimensional structure of AS1051 in a crystal.

FIG. 2 shows a reverse phase liquid chromatogram of AS1051-G4 afterlysyl endopeptidase digestion and structures of its peptide chains.

FIG. 3 shows a reverse phase liquid chromatogram ofpolyethylene-glycolated AS1051 (AS1051-PEG5000) after lysylendopeptidase digestion and structures of its peptide chains.

FIG. 4 shows inhibition curves of the polyethylene-glycolated AS1051 andmutant M23 proteins.

FIG. 5 shows inhibition for ristocetin-induced platelet aggregation bypolyethylene-glycolated M23.

FIG. 6 shows inhibition for thrombin-induced platelet aggregation by thepolyethylene-glycolated AS1051.

FIG. 7 shows numbers of platelets in guinea pigs after repetitiveadministrations of AS1051-Ala.

FIG. 8 shows results of detection of anti-AS1051-Ala antibodies inplasma of guinea pigs after repetitive administrations of AS1051-Ala.

FIG. 9 shows numbers of platelets in guinea pigs after repetitiveadministrations of AS1051-G4 and AS1051-PEG5000.

FIG. 10 shows ex vivo drug efficacy of M23-PEG20000, in which a)represents results of 5 minutes after the administration and b)represents results of 5 days after the administration.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described in detail below. In thefollowing description, an amino acid residue may be referred to by usinga three-letter code. When a numeral is added to the three-letter code,the numeral represents an amino acid number in the amino acid sequenceof SEQ ID NO: 1, unless otherwise specified. When a three-letter code isfurther added, it means replacement with an amino acid residue of thefurther added three-letter code (for example, “Cys81Ala” means that acysteine residue of the amino acid number 81 in the amino acid sequenceof SEQ ID NO: 1 is replaced with an alanine residue).

<1> Production Method of Present Invention and Protein of PresentInvention

The production method of the present invention comprises replacing, in aprotein that has an amino acid sequence having a homology of not lessthan 30% to the amino acid sequence of SEQ ID NO: 1 and forms a higherorder structure composed of a first β strand (β1), a first α helix (α1),a second α helix (α2), a second β strand (β2), a loop, a third β strand(β3), a fourth β strand (β4) and a fifth β strand (β5) in this orderfrom the amino terminus, at least one amino acid residue in a regionfrom α2 to β2 and/or a region from β3 to β4 so that electric charge ofthe amino acid residue is changed towards positive direction.

The present invention was accomplished based on the following findingsabout the binding scheme of AS1051 to glycoprotein Ib on the basis ofthe crystal structure of AS1051 elucidated in the present invention.

Observation of the crystal structure of AS1051 revealed that a regionforming two of α helixes (α1 and α2) and a region forming five β strands(β1 to β5) were recognized from the N-terminus and that a regioncomposed of β2, β3, β4 and α2 contained a large number of hydrophobicresidues and basic residues exposed to a solvent. Therefore, singleresidue mutants in which hydrophobic residues, basic residues and acidicresidues located in this region were replaced with Ala were prepared andtheir ability to bind to glycoprotein Ib was evaluated. The acidicresidues were also replaced aiming at suppressing repulsion to anegatively charged region of Tyr276 to Glu282 in glycoprotein Ib byweakening the negative electric charge of AS1051 and enhancing affinitybetween them. The mutation was introduced into a protein so that asequence between Val74 to Phe93 should be replaced with four Glyresidues (AS1051-G4). The abilities of the prepared 14 mutants to bindto glycoprotein Ib are shown in Example 5 (Table 2). The mutant in whichTyr58, Lys61, Tyr67, Arg103, Arg105 or Phe108 was replaced with Alashowed a markedly decreased binding ability, while the mutant in whichAsp54, Asp101 or Glu106 was replaced with Ala showed an increasedbinding ability.

Thus, it was revealed that the region containing these residues was abinding site for glycoprotein Ib. Further, it was suggested that thehydrophobic residues and the basic residues were involved in the bindingto glycoprotein Ib and that the acidic residues repelled the negativeelectric charge of glycoprotein Ib.

Subsequently, six mutants shown in Example 6 (Table 3) were preparedsince it was considered that the ability to bind to glycoprotein Ibcould be further improved by simultaneously replacing Asp54, Asp101 andGlu106. To eliminate the negative electric charge of the acidicresidues, replacement with Ala, Asn (in the case of Asp) or Gln (in thecase of Glu) was used in combination. These replacements were used sinceimprovement of the binding ability by single residue replacement withAla was confirmed and Asn and Gln change only electric charge withoutchanging the size. Among the prepared mutants, the Asp54Asn/Asp101Alamutant showed a higher glycoprotein Ib-binding ability than that of theAsp101Ala mutant.

It is known that glycoprotein Ib is composed of an α-chain and aβ-chain, and a domain binding to von Willebrand factor is in theN-terminus region of the α-chain (Titani et al., Proc. Nati. Acad. Sci.USA., 70, pp.538-542, 1987). This region is composed of 299 residues andis bonded with few saccharide chains. A region of Hisl to Leu275contains repeated sequences rich in Leu (leucine-rich repeats (Leu-richrepeats)) and has very high hydrophobicity. It has been reported thatboth of single residue replacements of Leu57 with Phe and Ala156 withVal in the Leu-rich repeats markedly decrease the binding ofglycoprotein Ib to von Willebrand factor (Miller et aL, Blood, 79,pp.439-446, 1992; Ware et al., J. Clin. Invest., 92, pp.1213-1220,1993). Further, a region of Tyr276 to Glu282 contains four acidicresidues and three sulfated Tyr residues, and it has been reported thatthis region is involved in the binding to vWF (von Willebrand factor;Ward et al., Biochemistry, 35, pp.4929-4938, 1996). These reportssupport the above findings.

From the above, it is considered that, as for a protein that hasantithrombotic activity similar to that of AS1051 and has such an aminoacid sequence containing a region from α2 to β2 and a region from β3 toβ4 in that order as in AS1051, its ability to bind to glycoprotein Ibcan be improved by replacing an amino acid residue located in the regionfrom α2 to β2 and/or the region from β3 to β4, which is considered to beimportant for the binding to glycoprotein Ib, with such an amino acidresidue that changes electric charge towards positive direction.

Examples of the protein having antithrombotic activity similar to thatof AS1051 include heterodimer proteins that bind to glycoprotein Iboriginating from snake venom, for example, glycoprotein Ib-bindingproteins originating from snake venom that have the same heterodimericstructure as that of CHH-B, wherein 30% or more of the amino acidsequences are homologous with that of the α-chain (AS1051) of CHH-Boriginating from Crotalus horridus horridus. In addition to the CHH-Bα-chain, for example, it has been indicated that a glycoproteinIb-binding activity is present in Echicetin (M. Peng et al., Biochem.Biophys. Res. Commun., 205, pp.68-72, 1994) and the β-chain ofJararaca-GPIb-BP (T. Kawasaki et al., J. Biol. Chem., 271,pp.10635-10639, 1996). The region from α2 to β2 and the region from β3to β4 of these proteins can also be identified by alignment of aminoacid sequences or alignment of the three-dimensional structures(threading). Further, subunits having a glycoprotein Ib-binding activityof other heterodimer glycoprotein Ib-binding proteins originating fromsnake venom are likewise applicable. A position of a subject structurefor designing an improved protein in a subunit of a protein of interestcan be identified by using a method such as X-ray crystallography or NMRof the protein. However, as shown in the present specification, it canalso be attained by estimating a subject partial structure in AS1051, apartial structure existing at positions corresponding to amino acidresidues, or the amino acid residues through alignment of amino acidsequence with the three-dimensional structure of AS1051 or alignment ofthe three-dimensional structures (threading). The alignment of aminoacid sequences can be performed by using a program of BLAST or the like.As for BLAST, a file compatible with a computer to be used can beobtained from files existing in ncbi.nlm.nih.gov/blast/excutable byusing FTP. Further, the threading can also be performed by using aprogram of INSIGHT II, COMPASS or the like. INSIGHT II and COMPASS arecommercially available from MSI and Protein Engineering ResearchInstitute, respectively.

In the mutant AS1051, all the residues of which replacement with Alamarkedly decrease the activity, that is, amino acid residues that areconsidered to be important for binding to glycoprotein Ib, Tyr58, Lys61,Tyr67, Arg103, Arg105 and Phe108, exist in the region from α2 to β2(Ala47 to Leu72) and the region from β3 to β4 (Glu94 to Asp111) judgingfrom their crystal structures. When the above alignment (threading)method is applied to, for example, the β-chain of echicetin, which hasbeen reported to bind to glycoprotein Ib (M. Peng et al., Biochem.Biophys. Res. Commun., 205, pp.68-72, 1993; EMBL/GenBank accessionnumber: P81996), the result of BLAST shows that sequences correspondingto a sequence considered to be the binding region of the above AS1051 toglycoprotein Ib exist between Ser45 and Leu68 and between His95 andLys110, while the result of COMPASS shows that they exist between Ser45and Asp70 and between Glu93 and Lys110. Since eight basic amino acidresidues (Arg43, Lys46, Lys60, Arg94, Lys100, Arg108, Arg109, Lys110)exist in this region, these results support that this region correspondsto the region in AS1051 that is important for the binding toglycoprotein Ib. Therefore, as in the case of AS1051, the presentinvention also includes a method of increasing the activity by changingelectric charge of an amino acid residue having an a carbon atom within10 Å from the α carbon in these basic amino acids towards positivedirection. Since acidic amino acid residues (Glu47, Asp49, Glu62, Asp70,Glu93, Asp103) exist in this region, it is considered to be preferableto replace at least one of these amino acid residues with a neutralamino acid residue. Further, a similar method is applicable to otherglycoprotein Ib-binding proteins originating from snake venom, which arehomologous to AS1051.

Proteins having antithrombotic activity similar to that of AS1051contain an amino acid sequence having a homology of, usually not lessthan 20%, preferably not less than 30%, to the amino acid sequence ofSEQ ID NO: 1. Therefore, a protein to be modified can be easily selectedin this range. The values of homology to the amino acid sequence of SEQID NO: 1 are those calculated by the aforementioned method using BLAST.

Regions forming α helixes and β strands can be identified by using anestimation method known to those skilled in the art.

Replacement with an amino acid residue that can change electric chargetowards positive direction include replacement of an acidic amino acidresidue with a neutral or basic amino acid residue and replacement of aneutral amino acid residue with a basic amino acid residue. Preferred isreplacement of an acidic amino acid residue with a neutral amino acidresidue. A plurality of amino acid residues may be replaced.

Examples of the acidic amino acid residue include Asp, Glu and so forth.Examples of the neutral amino acid residue include Gly, Ser, Thr, Cys,Tyr, Asn, Gln, Ala, Val, Leu, Ile, Met, Trp, Phe, Pro and so forth.Examples of the basic amino acid residue include Arg, His, Lys and soforth.

Specific examples of the proteins having antithrombotic activity similarto that of AS1051 include those containing an amino acid sequence of thefollowing (A) or (B):

(A) an amino acid sequence of the amino acid numbers 47 to 111 in theamino acid sequence of SEQ ID NO: 1;

(B) the amino acid sequence according to (A), in which Cys of the aminoacid number 81 in the amino acid sequence of SEQ ID NO: 1 is replacedwith Ala.

In those proteins, the region from α2 to β2 is a region corresponding tothe amino acid numbers 47 to 72 in the amino acid sequence of SEQ ID NO:1 and the region from β3 to β4 is a region corresponding to the aminoacid numbers 94 to 111 in the amino acid sequence of SEQ ID NO: 1. Inthese proteins, an acidic amino acid residue having an a carbon atomwithin 10 Å from the α carbon atom in Arg of the amino acid number 103in the amino acid sequence of SEQ ID NO: 1 is preferably replaced with aneutral amino acid residue. Alternatively, it is preferred that anacidic amino acid residue within amino acid numbers 47 to 59 and 100 to106 in the amino acid sequence of SEQ ID NO: 1 is replaced with aneutral amino acid residue. Further, it is more preferred that theacidic amino acid residue is composed of one or more of residuesselected from Asp of the amino acid number 54, Asp of the amino acidnumber 101 and Glu of the amino acid number 106 in the amino acidsequence of SEQ ID NO: 1. The distance between αcarbons is determined byX-ray crystallography of the protein.

The production method of the present invention may comprise removal ofthe loop structure region that is not involved in the glycoproteinIb-binding activity. That is, a region containing the loop structureexisting between β2 and β3 may be deleted in such a manner that thehigher order structures of β2 and β3 are maintained, or replaced withone or more amino acid residue(s) required to maintain the higher orderstructures of β2 and β3, which amino acid residue(s) are selected fromthe group consisting of Gly, Ala, Ser and Cys. It is preferred that aregion containing the loop structure is replaced with an amino acidsequence composed of four Gly residues.

This embodiment of the present invention was accomplished based on thefollowing findings about the crystal structure of AS1051 elucidated inthe present invention.

Based on the crystal structure, it was revealed that a region in thevicinity of Cys81 (crystal was prepared as for the protein containingthe mutation of Cys81Ala) involved in formation of dimer of AS1051,which originally exists as a heterodimer, had a flexible loop structurewith high hydrophobicity. In the heterodimer protein existing in snakevenom, CHH-B, the AS1051 moiety, i.e., the α-chain, binds to the β-chainvia Cys81. In consideration that the loop structure found in the presentinvention was important for this dimer formation and was not involved inthe binding to glycoprotein Ib, a protein in which a sequence from Val74to Phe93 was replaced with four Gly residues (AS1051-G4) was designed inorder to remove the region containing the loop structure withoutchanging the whole structure of the protein.

It was elucidated by preparing a protein AS1051-G4 and determining itsinhibitory activity for binding of AS1051 to glycoprotein Ib and vonWillebrand factor that the region containing the flexible loop structurefrom Val74 to Phe93 was important for the dimer formation, but was notinvolved in the binding to glycoprotein Ib. That is, the preparedAS1051-G4 exhibited almost the same inhibitory activity as that ofAS1051. It was considered that such a loop structure with highhydrophobicity was easily recognized as an antigen (immunodominant),deteriorated stability of the protein in a solution and so forth. As aresult of repetitive administrations of AS1051-G4 to animals in the samemanner as performed with AS1051, it was suggested that itsplatelet-reducing action was weaker and hence it had decreasedantigenicity. Further, a protein in which a peptide chain having abinding activity is maintained and a region not involved in the activityis replaced with Gly to decrease antigenicity is also obtained in, forexample, transformation of a protein originating leech saliva, hirudin,to hirulog (G. F. Pineo and R. D. Hull, Curr. Opin. Hematol., 2,pp.380-5, 1995). Therefore, the finding of a partial structure notinvolved in such an activity in a subunit of a glycoprotein Ib-bindingprotein originating from snake venom and the finding that this partialstructure can be replaced with a Gly chain are considered to beapplicable to other glycoprotein Ib-binding proteins originating fromsnake venom having a homologous amino acid sequence.

In AS1051, for example, the region containing the loop structure betweenβ2 and β3 corresponds to a sequence from Val74 to Phe93, and the numberof residues required to maintain the higher order structure of β2 and β3is four residues. However, the starting position and the end position ofthe partial sequence to be removed may be moved backward or forwardabout 10 residues, preferably about 3 to 5 residues. Further, the lengthof Gly chain to be inserted instead can be a length that provides themost desired activity in the range of 0 to 10 residues. Further, insteadof Gly, amino acid residues showing low hydrophobicity such as Ala, Serand Cys may also be used.

Specific examples of the protein in which a region containing the loopstructure is removed include a protein in which a sequence from Val74 toPhe93 in AS1051 is replaced with four Gly residues (AS1051-G4) and aprotein in which Met corresponding to the translation initiation pointis added to its amino terminus.

The loop structure region not involved in the glycoprotein Ib-bindingactivity in proteins other than AS1051 and the required number ofresidues can be identified based on comparison of their amino acidsequences with that of AS1051.

According to this embodiment of the present invention, a protein thatmaintains an inhibitory activity for binding of glycoprotein Ib and vonWillebrand factor (glycoprotein Ib/von Willebrand factor bindinginhibitory activity) and has decreased antigenicity can be obtained.

Further, the improved proteins obtained as described above arepreferably modified with polyoxyalkylpolyol to attain (1) increasedhalf-life/drug efficacy retention in blood and (2) decreasedantigenicity. This embodiment of the present invention was accomplishedbased on the following findings.

It was observed that polyethylene-glycolation of an improved protein ofAS1051 decreased or eliminated antigenicity observed upon theadministration of AS1051 to animals, and inhibited thrombin-inducedplatelet aggregation, which was not observed with AS1051. Thethrombin-induced aggregation inhibitory activity has been reported forsome glycoprotein Ib-binding proteins originating from snake venom, buthas not been recognized in AS1051 or CHH-B. Therefore, it is consideredthat their binding positions on glycoprotein Ib are different from thoseof the reported proteins (M. C. Chang et al., Blood, 91, pp.1582-9,1998). The glycoprotein Ib is considered to be one of platelet receptorsto thrombin (G. A. Jamieson, Thromb. Haemost., 78, pp.242-246, 1997),and it is also considered that inhibition of thrombin-induced plateletaggregation without affecting thrombin-induced coagulation reaction isan effective target of an antithrombotic drug (P. Andrew-Gordon et al.,Proc. Natl. Acad. Sci. USA., 96, pp.12257-12262, 1999). It is consideredthat the inhibitory activity on thrombin-induced aggregation provided bythe polyethylene-glycolation according to the present invention, notbecause the binding site on glycoprotein Ib was changed, but becausesteric hindrance by the polyethylene glycol region affected the bindingof thrombin and glycoprotein Ib.

Further, when a polyethylene-glycolated mutant having an increasedactivity was administered in a dose 10 times as much as the minimal drugefficacy concentration, retention of the inhibitory activity on plateletaggregation (botrocetin-induced aggregation) dependent on glycoproteinIb and von Willebrand factor, which is the drug efficacy, was observedeven 5 days (120 hours) after the administration.

Specific examples of polyoxyalkylpolyol include polyethylene glycol andso forth. Specific examples of its binding position include an aminogroup, a carboxyl group, a thiol group and so forth.

In this embodiment, it is preferred that the protein contains Cyscorresponding to Cys of the amino acid number 81 in the amino acidsequence of SEQ ID NO: 1 and a polyoxyalkylpolyol group is bonded tothis Cys. Further, in the case of a protein where the region containingthe loop structure between β2 and β3 is replaced, if Cys is contained inthe amino acid residue with which the loop structure is replaced, thepolyoxyalkylpolyol group may be bonded to the Cys.

Polyoxyalkylpolyolation is performed by, for example, the followingsteps (a) and (b).

(1) Step (a)

First, a protein or an oligomer thereof is denatured by using aprotein-denaturing agent in a solution. Then, the denaturing agent isremoved from the solution in the presence of polyoxyalkyl polyetherhaving a functional group that reacts with a thiol group to refold asubunit peptide.

A solvent of the solution is usually water. The protein-denaturing agentis not particularly limited so long as it can reversibly denature aprotein. Examples thereof include guanidine hydrochloride, urea and soforth. The protein-denaturing agent can be used at any concentration solong as the protein is dissolved. However, for example, it can be usedin the range of from 1 M to a saturated concentration, preferably, from2 M to 8 M. pH of the solution is not limited, but it is preferably inthe range of from 7 to 12, in which cleavage of a disulfide bond andbinding of polyethylene glycol to a thiol group, which will be explainedlater, readily occur. The temperature of the solution is not alsoparticularly limited, but it is preferably in the range from 0 to 40° C.The reaction time is also appropriately selected. The denaturation maybe performed under either reducing conditions or non-reducingconditions.

A disulfide bond in a protein or an oligomer thereof may be cleavedbeforehand by using a reducing agent, but this is not essential. Asubstance containing Cys such as glutathione, a reducing agent such asdithiothreitol, an enzyme such as a protein disulfide isomerase or thelike can be added before the refolding process.

When a protein or an oligomer thereof has a disulfide bond betweenproteins or a disulfide bond within the protein which is different fromthe disulfide bond in the original protein, the denaturation ispreferably performed under reducing conditions. In the presentinvention, the reducing conditions means conditions under which cleavageof a disulfide bond is promoted as in the presence of a substancecontaining Cys, a reducing agent, protein disulfide isomerase or thelike. Since cleavage of the disulfide bond is promoted under reducingconditions, the reaction with polyoxyalkyl polyether having a functionalgroup that reacts with a thiol group is promoted in the refoldingprocess. When an oligomer is denatured, it is particularly preferredthat the denaturation is performed under reducing conditions becausecleavage of a disulfide bond between proteins is promoted.

Subsequently, the denaturing agent is removed from the solutioncontaining the denatured protein in the presence of polyoxyalkylpolyether having a functional group that reacts with a thiol group. Thedenaturing agent can be removed from the solution by, for example,dialysis.

Typical examples of the functional group that reacts with a thiol groupinclude a maleimide group (R. J. Goodson et al., Bio/Technology, 8,p.343, 1990), orthopyridyl disulfide group (M. Yokoyama et al., Biochem.Biophys. Res. Commun., 164, p.1234, 1989), vinylsulfone group(Shearwater Polymers Inc. Item No. M-VS-5000) and so forth. Thefunctional group is not limited so long as it preferentially bonds to athiol group. Examples of the polyoxyalkyl polyether include polyethyleneglycol, polypropylene glycol, polyhydroxyethyl glycerol, dextran,carbohydrate polymers and so forth. The molecular weight is notparticularly limited, but it is preferably in the range of from 1000 to1,000,000, more preferably in the range of from 2000 to 50,000, in viewof improvement of solubility and decrease of antigenicity of a proteinto be obtained, and reactivity with a protein.

The polyoxyalkyl polyether described above may be added to the solutionbefore or after denaturation of a protein or an oligomer thereof. It mayalso be added before removing the denaturing agent. Usually, it ispreferred that after a protein or an oligomer thereof is denatured witha denaturing agent, the polyoxyalkyl polyether is added and the mixtureis allowed to react for a certain period of time and then the denaturingagent is removed from the solution. The amount of polyoxyalkyl polyetheris preferably an equimolar amount or more to the amount of the proteinto be reacted.

The polyoxyalkyl polyether bonds to Cys of a protein by allowing theprotein to react with polyoxyalkyl polyether having a functional groupthat reacts with a thiol group during or after the denaturation.

When the denaturing agent is removed from the solution as describedabove, refolding of the denatured protein occurs, and thus a peptidehaving a physiological activity identical to the physiological activityof the protein before modification or an activity of inhibiting thephysiological activity can be obtained.

By performing natural oxidation (air oxidation) to form a disulfide bondwithin a protein and then adding polyoxyalkyl polyether having afunctional group that reacts with a thiol group before the step ofremoving the denaturing agent from the solution containing the denaturedprotein, a polyoxyalkyl polyether group can be selectively andefficiently bonded to Cys originally involved in formation of adisulfide bond between subunits of the oligomeric protein.

(2) Step (b)

The protein bonded to polyoxyalkyl polyether, which is produced asdescribed above, is isolated from the solution. This operation can beperformed by a combination of operations used for conventionalpurification of a protein, that is, widely used chromatographytechniques such as ion exchange, gel filtration or reverse phasechromatography, electrophoresis, precipitation operation such as saltingout, desalting operation, concentration operation and so forth.

An aimed protein and polyoxyalkyl polyether can be separated by theabove operations. When an oligomeric protein is used as a startmaterial, the aimed protein and other proteins can be separated. Forexample, a protein having decreased antigenicity in which polyoxyalkylpolyether is bonded to Cys originally involved in formation of adisulfide bond between proteins in an oligomeric protein among Cys inthe protein can be separated from the other proteins.

The binding position of polyoxyalkyl polyether bonded to a protein ispreferably at Cys that forms a disulfide bond between proteins in theoligomeric protein. However, when the formation of the disulfide bondsis not determined, it may be such a position that the polyoxyalkylpolyether bonds to a specific thiol group, whereby the protein has anaimed activity, stably exists in a solution and preferably has decreasedantigenicity. The other disulfide bonds within the protein arepreferably the disulfide bonds within the protein of the originaloligomer. However, they can be different from them in such a degree thatthe protein still has a substantial physiological activity. Further,when the original disulfide bond within the protein is not determined,it may be such a position that the protein is identifiable as a singlemolecule having a physiological activity and stably exists in asolution. The number of polyoxyalkyl polyether molecules to be bondedper molecule is preferably equal to the number of Cys forming adisulfide bond between proteins in the original dimer protein. However,when the number is not determined, it may be such a number that theobtained polyoxyalkyl-polyetherated monomer protein has a physiologicalactivity, be identifiable as a single molecule and stably exists in asolution.

In the present invention, the modification with polyethylene glycol notonly decreases antigenicity upon administration to animals, but alsoincreases the glycoprotein Ib/von Willebrand factor binding inhibitoryactivity. That is, by modifying a protein in which its activity isincreased through such replacement that electric charge of amino acidresidues in a specific region should be changed towards positivedirection according to the present invention, an improved protein can befinally obtained by polyethylene-glycolation, which has (1) a markedlyincreased glycoprotein Ib/von Willebrand factor binding inhibitoryactivity compared with a protein before the improvement and (2)extremely prolonged retention of drug efficacy (glycoprotein Ib/vonWillebrand factor inhibiting inhibitory activity) after administrationto animals.

A protein prepared by a combination of (1) such replacement thatelectric charge of amino acid residues in the aforementioned specificregion should be changed towards positive direction, (2) removal of theloop structure region not involved in the glycoprotein Ib-bindingactivity and (3) polyoxyalkylpolyolation maintains high drug efficacy(inhibitory effect on glycoprotein Ib/von Willebrand factor binding)even 5 days after the administration to animals and has a novel activitythat is not observed in the original protein, i.e., thrombin-inducedplatelet aggregation inhibitory action, and the antigenicity observed inthe original protein is removed.

The protein of the present invention is a protein that can be producedby the production method of the present invention.

In the case of AS1051, a residue of the amino acid number 81 in theamino acid sequence of SEQ ID NO: 1 may be Ala or Cys in a proteinbefore modification. Further, Met corresponding to the translationinitiation point may be added to an amino terminus. Further, an improvedAS1051 prepared by the aforementioned “removal of the loop structureregion not involved in the glycoprotein Ib-binding activity” can also beused.

The residues most desired for the amino acid replacement in the aminoacid sequence of SEQ ID NO: 1 are Asp101, Glu106 and Asp54. Thesemutations may be such replacements with an amino acid that electriccharge should be changed. However, Asp is preferably replaced with Ala,Ser or Asn, and Glu is preferably replaced with Ala, Ser or Gln so thatthe whole structure of the protein should not be greatly changed.Further, as for the number of the amino acids to be replaced, one of theaforementioned amino acids may be replaced or a plurality of them may bereplaced in combination.

More specifically, examples of the protein of the present inventioninclude AS1051 having the amino acid sequence of SEQ ID NO: 1, such aprotein in which its Cys81 is replaced with Ala and a protein wherein apart of the amino acid sequence is removed as in the above example, forexample, the aforementioned AS1051-G4, in which one or more amino acidresidues of Asp54, Asp101 and Glu106 (numerals are amino acid numberscorresponding to those in SEQ ID NO: 1 and, in a protein obtained bymodifying a part of SEQ ID NO: 1, numerals are amino acid numbersparallerized in consideration to the change of an amino acid number) arereplaced, i.e., Asp is replaced with one of amino acids having differentcharacteristics such as Ala, Ser and Asn, and Glu is replaced with oneof amino acids having different characteristics such as Ala, Ser andGln.

The scheme of the disulfide bonds in the molecule of the protein of thepresent invention is preferably the same as that of the original dimericprotein, CHH-B. However, since the scheme in a natural-type CHH-B hasnot been reported, it may be the same as, for example, that of AS1051having substantial physiological activity (N. Fukuchi et al.,WO95/08573) or it may be different in such a degree that thephysiological activity is not greatly lost. Further, when the disulfidebond in the original subunit is not determined, it is not particularlylimited so long as it can be identified as a single molecule having aphysiological activity and stably exists in a solution. Further, theamino acid sequences may include insertion, deletion, substitution andso forth in a part of amino acid residues so long as they do notsubstantially change the antithrombotic activity.

Further, properties of the protein obtained by the production method ofthe present invention are not particularly limited so long as theyinclude one or a combination of (1) an increased binding activity to atarget (glycoprotein Ib), (2) increased half-life/drug efficacyretention in blood, and (3) decreased antigenicity compared with aprotein before the improvement. The increased binding activityspecifically means that the Kd value for glycoprotein Ib becomes ½ orless or the IC₅₀ value of glycoprotein Ib/von Willebrand factor bindinginhibitory activity becomes ½ or less compared with those of a proteinbefore the improvement under the same measurement conditions. Further,the increased half-life/drug efficacy retention in blood is notparticularly limited so long as a significant increase of thehalf-life/drug efficacy retention in blood is observed compared with aprotein before the improvement. Where there are obtained no findingsabout the half-life/drug efficacy retention in blood of the proteinbefore the improvement, it is sufficient that drug efficacy retention of1 day (24 hours) or longer after administration should be observed inconsideration of general characteristics of an exogenous protein.Further, it is sufficient that the antigenicity should be substantiallydecreased compared with a protein before the improvement. Where thereare obtained no findings about antigenicity of the protein before theimprovement, it may be such antigenicity that biological and biochemicalreactions attributable to an antigen-antibody reaction should not occurwhen the obtained improved protein is administered to an animal in aminimal dose required to provide the obtained drug efficacy and in aminimal number of times required for immunization and then administeredagain.

Examples of the physiological activity of the protein obtained by theproduction method of the present invention include an inhibitory actionfor glycoprotein Ib-dependent platelet aggregation (platelet aggregationattributable to an inducing substance such as ristocetin or botrocetin)exerted by binding to glycoprotein Ib, antithrombotic action,anti-inflammatory action and analgesic action based on platelet adhesioninhibition and so forth.

The antithrombotic activity can be evaluated by a known method, forexample, a method for determining an inhibitory activity for binding ofglycoprotein Ib and von Willebrand factor, which will be described inthe examples later.

Replacement of an amino acid in the production method of the presentinvention can be performed by preparing a DNA coding for an amino acidsequence after replacement as described in the explanation about the DNAof the present invention mentioned later. A modified protein in which anamino acid is replaced can be prepared by culturing a host microorganismtransformed with that DNA and collecting a protein encoded by the DNAfrom a culture.

<2> DNA of Present Invention and Method for Producing Protein of PresentInvention Using the Same

The DNA of the present invention is a DNA coding for the protein of thepresent invention. The protein of the present invention can be preparedby culturing a host microorganism transformed with the DNA of thepresent invention and collecting a protein encoded by the DNA from aculture. Further, the polyoxyalkylpolyolated protein of the presentinvention can be produced by bonding a polyoxyalkylpolyol group to thecollected protein.

The DNA of the present invention can be prepared by obtaining DNA codingfor a protein before modification (target protein) and modifying thisDNA.

When the amino acid sequence of the protein or the full length or a partof the nucleotide sequence of DNA coding for the protein is known, theDNA coding for the target protein can be obtained by the polymerasechain reaction (PCR) method using primers prepared based on a knownnucleotide sequence. Alternatively, it can also be obtained from a cDNAlibrary by performing hybridization using a probe prepared based on aknown nucleotide sequence. Further, when DNA coding for the targetprotein is deposited at a depository, the deposited DNA can be used.Further, the whole nucleotide sequence of the gene is known, the DNA canbe chemically synthesized. For example, the E. coli HB101/pCHA1 (E. coliAJ13023) strain harboring a plasmid containing the gene sequence ofAS1051 was deposited at the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, Ministryof International Trade and Industry (postal code: 305-8566, 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki-ken, Japan) as an international depositionunder the provisions of the Budapest Treaty on Aug. 12, 1994, andreceived an accession number of FERM BP-4781.

DNA coding for the improved protein (mutant protein gene) can also beobtained by the PCR method using DNA coding for the target proteinobtained as described above as a template and primers prepared so as toinclude a mutated nucleotide sequence, or by cleaving the gene andreplacing it with a nucleotide sequence containing a mutation.

An expression system of the mutant protein gene obtained as describedabove using a microorganism can be constructed by incorporating the genecoding for the protein into a protein expression vector having apromoter that can be expressed in a commercially available host or ahost prepared by using, for example, Escherichia coli, Bacillussubtilis, yeast or the like, and the gene can be expressed. The proteinto be expressed can be directly expressed in the form having a signalsequence or in the form of a protein encoded by the gene coding for theprotein, to which a translation initiation codon, methionine is added.It can also be expressed as a fusion protein to which a maltose bindingprotein (MBP), glutathione S-transferase (GST), calmodulin bindingpeptide, thioredoxin, His-tag or the like is added. Examples of thevector for the former direct expression include vectors such as pET(stratagene), pGEMEX (Promega), pTrc99A (Amersham Pharmacia) and soforth. Examples of the vector for the latter expression as a fusionprotein include pMAL (New England Bio Lab) for an MBP fusion protein,pGEX (Amersham Pharmacia Biotech) for a GST fusion protein, pCAL(Stratagene) for a fusion protein with a calmodulin-binding peptide,pTrcFus (Invitrogen) for a fusion protein with thioredoxin and pTrcHis(Invitrogen) for a fusion protein with His-tag. Further, these vectorscan be appropriately modified and used. Further, as an expressionscheme, a method of accumulating the protein as a granule in microbialcells can be used, but the protein can also be accumulated or secretedas a soluble type protein.

Further, an expression system using cells of the prepared mutant proteingene can be constructed by using animal cells, insect cells or the likeas a host and incorporating the gene coding for the protein into avector having a promoter that can be expressed in the host to attainexpression.

Refolding of a protein obtained in a form not having the activity can beperformed as follows, in which the protein is converted into a formhaving the activity by cross-linking disulfide bonds in a molecule.First, the granular or soluble type protein that does not have theactivity is dissolved in a solution containing a protein-denaturingagent such as guanidine hydrochloride or urea. The protein-denaturingagent can be used at any concentration so long as the protein isdissolved. However, for example, it can be used in the range of from 1 Mto a saturated concentration, preferably, from 2 M to 8 M. pH of thesolution is not limited, but it is preferably in the range of from 7 to12, in which cleavage and bonding of a disulfide bond readily occur. Thetemperature of the solution is not also particularly limited, but it ispreferably in the range from 0 to 40° C. A disulfide bond of the proteinmay be cleaved beforehand by using a reducing agent, but this is notessential. A substance containing Cys such as glutathione, a reducingagent such as dithiothreitol, an enzyme such as a protein disulfideisomerase or the like can also be added during the refolding process.

Further, in order to modify the obtained protein withpolyoxyalkylpolyol, a polyoxyalkylpolyol that reacts with an aminogroup, carboxyl group, thiol group or the like can be added so that itbinds to the protein after or during the refolding of the protein asdescribed above. As a specific example of the polyoxyalkylpolyol,polyethylene glycol can be mentioned. Specific examples of the methodfor binding it to a protein include a method of reacting with a thiolgroup of Cys.

The above protein having the activity produced by bacteria or cells orthe above protein converted into a form of having the activity byrefolding can be purified by a combination of widely used chromatographysuch as ion exchange, gel filtration or reverse phase chromatography,electrophoresis, precipitation operation such as salting out, desaltingoperation, concentration operation and so forth.

<3> Drug Comprising Protein of Present Invention as Active Ingredient

The protein of the present invention can be utilized as anantithrombotic drug. Since the protein of the present invention isimparted with improvements such as (1) an increased binding activity toits target (glycoprotein Ib), (2) increased half-life/drug efficacyretention in blood and (3) decreased antigenicity compared with aprotein before the improvement, it can be used as an anti-inflammatorydrug and analgesic drug based on platelet adhesion in addition to a moreeffective antithrombotic drug.

The protein of the present invention in a drug comprising the protein ofthe present invention as an active ingredient may be used as it is or asa pharmaceutically acceptable salt thereof. The protein can be used aseach alone or as a mixture of two or more kinds of the protein. Otheractive ingredients may also be added. Usually, it may be mixed withother materials for use in conventional pharmaceutical preparations, forexample, ingredients including proteins such as serum albumin,surfactants, salts for a buffering action and osmotic adjustment,carriers and excipients to prepare a pharmaceutical composition.

Examples of the dosage form include a tablet, capsule, subtilizedgranule, syrup, suppository, ointment, injection, eye drop and so forth.Among these, injection is preferred. As the administration method, anyof methods such as intravenous administration, subcutaneousadministration, intramuscular administration, oral administration,instillation and enteral administration can be used, but intravenousadministration, subcutaneous administration, intramuscularadministration and so forth are preferred among them.

As for dose for an animal or human, a dose in the range from 0.1 μg/kgto 100 mg/kg as the amount of the protein of the present invention canusually be expected to provide the desired effect, and a dose that canprovide the optimal drug efficacy can be selected within this range.

EXAMPLES

Hereafter, the present invention will be explained in more detail withreference to the following examples.

Example 1

Determination of Three-dimensional Structure of CHH-B α-chain Protein(AS1051-Ala)

(1) Construction of CHH-B α-chain Protein (AS1051-Ala) Expression SystemUsing Escherichia coli

An expression system using Escherichia coli for the CHH-B α-chainprotein wherein the Cys residue in position 81 was mutated to Ala(AS1051-Ala) was constructed by using the E. coli HB101/pCHA1 strain (E.coli AJ13023) harboring pCHA1 (deposited at the National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology, Ministry of International Trade and Industry (postal code:305-8566, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) as aninternational deposition under the provisions of the Budapest Treaty onAug. 12, 1994 and given an accession number of FERM BP-4781) as follows.The nucleotide sequence of the gene coding for the protein contained inpCHA1 that originates in Crotalus horridus horridus and contains AS1051(its amino acid sequence is shown as SEQ ID NO: 1 in Sequence Listing)is shown as SEQ ID NO: 2 and the amino acid sequence of the encodedprotein is shown as SEQ ID NO: 3.

First, a mutation was introduced into the AS1051 gene by thesite-specific nucleotide sequence mutation method described in PCRProtocols (Academic Press edition) as follows so that a Cys residue (Cysin position 81 in SEQ ID NO: 1) not involved in a disulfide bond in theAS1051 peptide should be replaced with Ala. PCR was performed by usingpCHA1 as a template and primers ASBN (SEQ ID NO: 4) and ASAlaR (SEQ IDNO: 5) or primers ASH (SEQ ID NO: 6) and ASAlaF (SEQ ID NO: 7). Eachreaction product was subjected to agarose gel electrophoresis and anamplified DNA fragment was extracted from the gel. The second PCR wasperformed by using each DNA fragment as a template and the primers ASBNand ASH to prepare a mutant gene. The PCR-amplified DNA was subjected toagarose gel electrophoresis and DNA of 400 base pairs was extracted fromthe gel. This DNA was digested with restriction enzymes BamHI andHindIII. This DNA fragment was ligated with a plasmid pUC18 (TakaraShuzo) digested with the restriction enzymes BamHI and HindIII by usinga Ligation Kit (Takara Shuzo). The E. coli JM109 strain was transformedwith the obtained plasmid by the competent cell method and cultured onan ampicillin-containing plate at 37° C. for 16 hours to select atransformant.

A plasmid was prepared from the obtained transformant by the alkalineSDS method. Construction of the target plasmid was confirmed bydetermining the nucleotide sequence by using M13M4 and M13RV primers(both from Takara Shuzo) and a 377PRISM DNA sequencer (Perkin-Elmer).The prepared plasmid was designated as pUCASAla. The plasmid pUCASAlawas digested with restriction enzymes NcoI and HindIII and subjected toagarose gel electrophoresis to separate and purify DNA of 400 basepairs. This DNA was ligated with a product obtained by digesting anexpression vector pTrcHisA (Invitrogen) with the restriction enzymesNcoI and HindIII by using the Ligation Kit. The E. coli JM109 strain wastransformed with the obtained plasmid by the competent cell method andcultured on an ampicillin-containing plate at 37° C. for 16 hours toselect a transformant. The expression vector contained in thistransformant was designated as pTrcASAla.

(2) Preparation of Active Substance by Refolding of AS1051-Ala

The transformant E. coli JM109 strain harboring the expression vectorpTrcASAla for AS1051-Ala was cultured at 37° C. in L-broth (1% Bactotrypton, 0.5% yeast extract, 0.5% sodium chloride, 100 μg/ml ampicillinsodium) by using a Sakaguchi flask. IPTG(isopropyl-β-thiogalactopyranoside) was added at 10 mM when theturbidity (measured at 660 nm, ditto for the following examples) reached0.5 and further cultured at 37° C. for 4 hours. Microbial cells werecollected and washed by centrifugation. Then, the microbial cells weresuspended in 0.5 M EDTA solution. Lysozyme was added thereto and themixture was then left standing at room temperature for 1 hour. Thesuspension of the microbial cells was disrupted by an ultrasonicator(200 W, 5 minutes), and the disrupted suspension was centrifuged toobtain granules (inclusion bodies) as precipitates.

The obtained granules were dissolved in 0.5 M Tris-HCl buffer (pH 8.5)containing 7 M guanidine hydrochloride and 10 mM EDTA. Then, distilledwater was added in a volume of 2.5 times the solution and the mixturewas left standing overnight at 4° C. The solution was dialyzed against0.9% saline by using a Spectra/Por 1 dialysis membrane (Spectra) toremove guanidine hydrochloride. To the solution after the dialysis, 1/9volume of 0.5 M ammonium acetate buffer (pH 4.5) was added, and themixture was loaded on an ion exchange column using CM-TOYOPEARL 650S(2.6×40 cm) and eluted with Elution solution A (50 mM ammonium acetatebuffer (pH 4.5)) and Elution solution B (0.5 M ammonium acetate buffer(pH 6.4)). The elution was performed with a solution composed of Elutionsolution A and B (A:B=75:25, volume ratio, ditto in the followingexamples) for 20 minutes, and then with a linear gradient of fromA:B=75:25 to A:B=50:50 for 30 minutes. Thus, an eluted fractioncontaining purified AS1051-Ala was obtained.

AS1051-Ala has a structure in which Met is added to the N-terminus ofthe amino acid sequence of SEQ ID NO: 1 and Cys81 is replaced with Ala.

(3) Determination of Three-dimensional Structure of AS1051-Ala by X-rayCrystal Structure Analysis

The purified AS1051-Ala was crystallized by the vapor diffusion methodin sitting drops method as follows. A mixed solution of 5 μl each of abuffer (reservoir solution) composed of 0.1 M NaHepes (pH 7.3), 12%PEG4K (polyethylene glycol having a molecular weight of 4000), 22%2-propanol, 10 mM CaCl₂ and 10 mg/ml of AS1051-Ala solution was placedin a cavity of a bridge provided over a well filled with 500 μl of thereservoir solution and left standing in the sealed well at 20° C.Tabular crystals in the maximum size of 0.8×0.2×0.15 mm were obtained inabout 10 days to 2 weeks. To obtain X-ray diffraction data, the crystalswere transferred into a buffer containing 0.1 M NaHepes (pH 7.3), 12%PEG4K and 22% 2-propanol. X-ray diffraction data up to 2.5 Å resolutionwere collected at room temperature by using an X-ray diffractometerR-AXIS IIc (Rigaku Corporation) to determine crystallographicparameters. The space group was β2₁2₁2, and the cell parameters werea=44.7 Å, b=66.7 Å and c=46.8 Å. The solvent content of the crystals wasestimated to be 47%. Further, 1.8 Å resolution X-ray data were collectedby using a Weissenberg camera for macromolecules installed in BL6A of asynchrotron radiation facility in the High Energy Physics Laboratory.

The crystal structure analysis was performed by the heavy atomisomorphous replacement method. The crystals were immersed in 1 to 10 mMsolutions of heavy metal salts, and X-ray data were measured by usingthe R-AXIS IIc to screen heavy atom isomorphous crystals. As a result,it was revealed that X-ray data of the crystals immersed in solutions ofthree heavy atom salts, cis-Pt(ethylenediamine)Cl₂, cis-Pt(NH₃)₂Cl₂ andtrans-Pt(NH₃)₂Cl₂ showed significant differences from the X-ray data ofthe native crystals. In particular, the cis-Pt(ethylenediamine)Cl₂showed an easily interpretable difference Patterson map. The coordinatesof the sole mercury binding site of the cis-Pt(ethylenediamine)Cl₂ wereobtained by using a program RSPS. These coordinates were refined byusing program MLPHARE and used to calculate the phases. The mercurybinding sites of the two other heavy metal salts, i.e., cis-Pt(NH₃)₂Cl₂and trans-Pt(NH₃)₂Cl₂, were obtained by using these phases. The threeheavy atom parameters of the heavy atom isomorphous replaced crystalswere simultaneously refined by using MLPHARE, and then solventflattering and histogram matching were performed by using program DM toimprove the phases. An anomalous dispersion effect of thecis-Pt(ethylenediamine)Cl₂ was also used. The electron density map at aresolution of 3 Å calculated by using the phases improved as describedabove had good quality sufficient to easily identify α helices or βstrands.

The α carbon chains were constructed by using program QUANTA (MSI).First, a region from Leu2 to Leu72 and a region from Glu94 to Arg124were constructed. It was difficult to interpret the region from Arg73 toPhe93 at this stage. The side chains of Leu2 to Leu72 and Glu94 toArg124 were added and then refined by using program X-PLOR (MSI). Then,the model was corrected by using QUANTA and refined by using X-PLOR,which were repeated until the R value decreased to 0.304 at a resolutionof 3 Å. At this point, the phases calculated from the model and thephases calculated from the heavy atom isomorphous replaced crystals werecombined by using program SIGMAA. By using the (Fo-Fc) map obtained asdescribed above, the region from Arg73 to Phe93 could be constructed asa polyalanine model. Further, the side chains of this region were addedstepwise by alternately using QUANTA and X-PLOR. In this process, theresolution was increased up to 1.8 Å. When the resolution exceeded 2.5Å, data obtained by using R-AXIS IIc was switched to data obtained byusing synchrotron radiation. Further, the structure was corrected andwater molecules were included. Finally, a model with an R value of 0.187was obtained for data in a resolution range between 6 and 1.8 Å (FIG.1). The final model contained 123 residues of Leu2 to Arg124 and 53water molecules. N-terminal Met, Asp1, Pro125, Arg126, and side chainsof Arg73, Val74, Gln75, Glu87, Phe93 and Arg105 were not observed. Therms value, which is a value of deviation from the ideal bond length, was0.019 Å, while that of the bonding angle was 2.898 Å. Further, theaverage temperature factor of the main chain atom was 23 Å² while thatof the side chain atom was 28 Å². When a Ramachandran plot was createdby using program PROCHECK, it was shown that 96% of residues other thanGly were located in the most favored region and that 3% was located inthe additionally allowed region. The programs RSPS, MLPHARE, DM, SIGMAAand PROCHECK are available from CCP4 (CCP4, Acta Crystallogr. D, 50,760-763).

Example 2

Preparation of Loop Structure-deleted AS1051 Protein (AS1051-G4)

(1) Examination of Central Loop Structure

There exists a long loop projecting to a solvent between β2 and β3 asshown in FIG. 1. This loop was designated as a central loop. The averagetemperature factor of the α carbon atoms in the central loop containing20 residues of Val74 to Phe93 was 37.4 Å², which was markedly higherthan the average temperature factor of the α carbon atoms other thanthose of the central loop, 20.2 Å². This indicated that flexibility ofthe central loop was extremely high. The central loop includes Ala81, aresidue corresponding to Cys81 in the wild-type AS1051, which isinvolved in an intermolecular S—S bond with the CHH β-chain. Therefore,while the central loop is considered to play an important role information of a dimer with the β-chain, it is considered that, when theα-chain (AS1051) exists solely, a hydrophobic residue is exposed to thesolvent and easily recognized as an antigen. Therefore, to confirmwhether antigenicity can be decreased by removing the central loop, amutant deficient in the central loop was prepared. Since the distancebetween the α carbon atoms of Val74 and Phe93 was 16.1 Å, it wasconsidered that the central loop could be replaced with a peptide offour residues. As the four amino acid residues, it was consideredappropriate to use glycine, which does not have a β carbon atom and haslittle restriction by a dihedral angle (φ/ψ value). The central loop wasreplaced with four Gly residues (G4) by using the program QUANTA (MSI),and the obtained structure was optimized by molecular dynamicscalculation by using the program X-PLOR (MSI). At this time, arestriction in the X-ray term was applied so that the structure otherthan G4 should not be significantly changed. When a Ramachandran plot ofthe optimized structure was created by using the program PROCHECK, itwas demonstrated that the four Gly residues and residues before andafter them were located in the most preferable region. That is, it wasindicated that the mutant (AS1051-G4) in which the central loop isreplaced with the four Gly residues could have a structure withoutdistortion.

(2) Construction of Expression Vector of AS1051-G4 for Escherichia coli

The gene coding for a mutant peptide AS1051-G4 having the amino acidsequence of SEQ ID NO: 1 of the AS1051 peptide wherein 20 amino acidsfrom the 74th Val to the 93rd Phe were replaced with four Gly residueswas prepared by using the cloned gene. DNA primers for preparing themutant gene were synthesized by the PCR method. As primers containingthe mutated region, G4F (SEQ ID NO: 8) and its complementary sequence,G4R (SEQ ID NO: 9), were prepared. Further, a primer containing an NcoIrecognition sequence (ASBN: SEQ ID NO: 4) was used as the 5′ end primerso that the 5′ end of the amplified fragment should have the NcoI siteand thus the mutant gene to be prepared could be incorporated into anexpression vector in the subsequent process. Further, this primer had anucleotide sequence ATG (nucleotide numbers 10 to 12 in SEQ ID NO: 4),which was a translation initiation codon, before a codon of theN-terminal amino acid AS1051 peptide, i.e., aspartic acid, on the 5′ endside. This initiation codon overlapped the NcoI recognition sequence(nucleotide numbers 8 to 13 in SEQ ID NO: 4). As the 3′ end primer, aprimer containing a HindIII recognition sequence (SEQ ID NO: 6, HindIIIrecognition sequence corresponds to nucleotide numbers 4 to 9) was used.First, PCR was performed by using pCAH1 as a template and the primersASBN and G4R with a cycle of reactions at 94° C. for 15 seconds, at 35°C. for 1 minute and at 72° C. for 2 minutes, which was repeated 25times. Similarly, PCR was performed by using pCAH1 as a template and theprimers ASH and G4R with a cycle of reactions at 94° C. for 15 seconds,at 35° C. for 1 minute and at 72° C. for 2 minutes, which was repeated25 times. Each PCR reaction mixture was subjected to agarose gelelectrophoresis. The amplified DNA fragments of 250 base pairs and 130base pairs were collected from the gel by using EASYTRAP (Takara Shuzo).As the second PCR, PCR was performed by using the DNA fragments of 250base pairs and 130 base pairs collected as described above and the ASBNprimer and ASH primer. PCR was performed with a cycle of reactions at94° C. for 15 seconds, at 35° C. for 1 minute and at 72° C. for 2minutes, which was repeated 25 times. The PCR reaction mixture wassubjected to a phenol/chloroform treatment to inactivate Taq polymerase.The amplified DNA fragment of 400 base pairs was purified by ethanolprecipitation and digested with restriction enzymes BamHI and HindIII.This DNA fragment was ligated with a plasmid pUC18 (Takara Shuzo)digested with restriction enzymes BamHI and HindIII by using a LigationKit (Takara Shuzo). The E. coli JM109 strain was transformed with theobtained plasmid by the competent cell method and cultured on anampicillin-containing plate at 37° C. for 16 hours to select atransformant. A plasmid was prepared from a grown transformant by thealkaline SDS method. Construction of the target plasmid was confirmed bydetermining the nucleotide sequence by using the M13M4 and M13RV primers(both from Takara Shuzo) and a 377PRISM DNA sequencer (Perkin-Elmer).The prepared mutant plasmid was designated as pUCASG4BNH.

The plasmid pUCASG4BNH was digested with restriction enzymes NcoI andHindIII and subjected to agarose gel electrophoresis to separate andpurify DNA of 350 base pairs. This DNA was ligated with a productobtained by digesting an expression vector pTrcHisA (Invitrogen) withrestriction enzymes NcoI and HindIII by using the Ligation Kit. The E.coli JM109 strain was transformed with the obtained plasmid by thecompetent cell method and cultured on an ampicillin-containing plate at37° C. for 16 hours to select a transformant. The expression vectorobtained as described above was designated as pTrcASG4. The expressedand produced AS1051-G4 peptide has a structure in which Met is added tothe N-terminus of the amino acid sequence of SEQ ID NO: 10.

(3) Acquisition of AS1051-G4

The transformant E. coli JM109 strain harboring the expression vectorpTrcASG4 for AS1051-G4 was cultured at 37° C. in L-broth (1% Bactotrypton, 0.5% yeast extract, 0.5% sodium chloride, 100 μg/ml ampicillinsodium) by using a Sakaguchi flask. IPTG(isopropyl-β-thiogalactopyranoside) was added at 10 mM when theturbidity reached 0.5 and further cultured at 37° C. for 4 hours. Themicrobial cells were collected and washed by centrifugation. Then, themicrobial cells were suspended in a 0.5 M EDTA solution. Lysozyme wasadded thereto and the mixture was left standing at room temperature for1 hour. The suspension of the microbial cells was disrupted by anultrasonicator (200 W, 5 minutes) and the disrupted suspension wascentrifuged to obtain granules (inclusion bodies) as precipitates.

The obtained granules were dissolved in 0.5 M Tris-HCl buffer (pH 8.5)containing 7 M guanidine hydrochloride and 10 mM EDTA. Then, distilledwater was added in a volume of 2.5 times the solution and the mixturewas left standing overnight at 4° C. The solution was dialyzed against0.9% saline by using a Spectra/Por 1 dialysis membrane (Spectra) toremove guanidine hydrochloride. To the solution after the dialysis, 1/9volume of 0.5 M ammonium acetate buffer (pH 4.5) was added, and themixture was loaded on an ion exchange column using CM-TOYOPEARL 650S(2.6×40 cm) and eluted with Elution solution A (50 mM ammonium acetatebuffer (pH 4.5)) and Elution solution B (0.5 M ammonium acetate buffer(pH 6.4)). The elution was performed with a solution composed of Elutionsolution A and B (A:B=70:30) for 20 minutes, and then with a lineargradient of from A:B=70:30 to A:B=40:60 for 30 minutes. Thus, an elutedfraction containing purified AS1051-G4 was obtained.

Example 3

Structure of Loop Structure-deleted AS1051 Protein (AS1051-G4)

SDS electrophoresis revealed that the molecular weight of the obtainedAS1051-G4 was 12 kDa, which was about 3 kDa lower than that of theaforementioned AS1051-Ala (15 kDa).

Subsequently, the linkage scheme of a disulfide bond of Cys in AS1051-G4was determined as follows. AS1051-G4 (100 μg) was digested with lysylendopeptidase (5 μg, Wako Pure Chemical Industries) in 0.1 M Tris-HClbuffer (pH 8.5) containing 2 mM EDTA at 37° C. for 5 hours andfractionated by high performance liquid chromatography using a reversephase column (Vydac 218TP54, Vydac). Elution was performed with a lineargradient of water/acetonitrile containing 0.1% trifluoroacetic acid(TFA) (acetonitrile concentration of from 0 to 50% for 10 minutes,acetonitrile concentration of from 50% to 100% for 5 minutes). Thus,digested peptide chains were obtained as Peaks 1 to 5 (FIG. 2). Theamino acid sequence of each peptide chain was analyzed by using aprotein sequencer Model 476A (Applied Biosystems). Since two Cysresidues are contained in the peptide chain of Peak 3, it was concludedthat these two Cys residues were coupled to form a disulfide bond.Further, it was found that Peak 4 was composed of total three peptidechains, wherein two peptide chains containing one Cys residue and onepeptide chain containing two Cys residues were coupled through disulfidebonds. The peptide chains of this Peak 4 were further digested with V8protease (5 μg, Wako Pure Chemical Industries) in 10 mM ammoniumcarbonate buffer at 25° C. for 24 hours and fractionated by highperformance liquid chromatography using a reverse phase column (PegasilODS-II, Senshu Kagaku). Elution was performed with a linear gradient ofwater/acetonitrile containing 0.1% TFA (acetonitrile concentration offrom 0 to 50% for 20 minutes). Thus, peptide chains obtained bydigestion were prepared and their amino acid sequences were analyzed. Asa result, it was found that the peptides of Peak 4 had such a disulfidebond as shown in FIG. 2. The scheme of disulfide bond in AS1051-G4determined as described above was the same as in the reported AS1051 (N.Fukuchi et al., WO95/08573) or other similar proteins originating fromsnake venom (Y. Fujimura et al., Thromb. Haemost., 76, pp.633-639(1996)).

Example 4

Preparation of Protein Containing Single Amino Acid Mutation

Proteins were prepared in which amino acids of AS1051-G4 prepared inExample 2 were mutated as shown in Table 1. The amino acid numbers usedin the table correspond to the amino acid numbers in SEQ ID NO: 1.

TABLE 1 Mutant protein Amino acid to be mutated Amino acid aftermutation M7-G4 Lys20 Ala20 M8-G4 Asp54 Ala54 M9-G4 Tyr58 Ala58 M10-G4Lys61 Ala61 M11-G4 Glu62 Ala62 M12-G4 Tyr63 Ala63 M13-G4 Arg66 Ala66M14-G4 Tyr67 Ala67 M15-G4 Arg100 Ala100 M16-G4 Asp101 Ala101 M17-G4Arg103 Ala103 M18-G4 Arg105 Ala105 M19-G4 Glu106 Ala106 M20-G4 Phe108Ala108

By using the expression plasmid pTrcASG4 for AS1051-G4 as a template andone of combinations of primers M7F (SEQ ID NO: 11) and M7R (SEQ ID NO:12), M8F (SEQ ID NO: 13) and M8R (SEQ ID NO: 14), M9F (SEQ ID NO: 15)and M9R (SEQ ID NO: 16), MIOF (SEQ ID NO: 17) and M10R (SEQ ID NO: 18),M11F (SEQ ID NO: 19) and M11R (SEQ ID NO: 20), M12F (SEQ ID NO: 21) andM12R (SEQ ID NO: 22), M13F (SEQ ID NO: 23) and M13R (SEQ ID NO: 24),M14F (SEQ ID NO: 25) and M14R (SEQ ID NO: 26), M15F (SEQ ID NO: 27) andM15R (SEQ ID NO: 28), M16F (SEQ ID NO: 29) and M16R (SEQ ID NO: 30),M17F (SEQ ID NO: 31) and M17R (SEQ ID NO: 32), M18F (SEQ ID NO: 33) andM18R (SEQ ID NO: 34), M19F (SEQ ID NO: 35) and M19R (SEQ ID NO: 36), andM20F (SEQ ID NO: 37) and M20R (SEQ ID NO: 38), PCR was performedaccording to the protocol attached to a QuickChange™ Site-DirectedMutagenesis Kit (Stratagene) to obtain plasmids pTrcM7G4, pTrcM8G4,pTrcM9G4, pTrcM10G4, pTrcM11G4, pTrcM12G4, pTrcM13G4, pTrcM14G4,pTrcM15G4, pTrcM16G4, pTrcM17G4, pTrcM18G4, pTrcM19G4 and pTrcM20G4expressing mutant proteins M7-G4, M8-G4, M9-G4, M10-G4, M11-G4, M12-G4,M13-G4, M14-G4, M15-G4, M16-G4, M17-G4, M18-G4, M19-G4 and M20-G4,respectively.

The transformant E. coli JM109 strains harboring the prepared expressionplasmids pTrcM8G4 to pTrcM20G4 were each cultured at 37° C. in L-broth(1% Bacto trypton, 0.5% yeast extract, 0.5% sodium chloride, 100 μg/mlampicillin sodium). IPTG (isopropyl-β-thiogalactopyranoside) was addedat 0.5 mM when the turbidity reached 0.5 and further cultured at 37° C.for 4 hours. The microbial cells were collected and washed bycentrifugation. Then, the microbial cells were suspended in a 0.5 M EDTAsolution. Lysozyme was added thereto and the mixture was left standingat room temperature for 1 hour. The suspension of the microbial cellswas disrupted by an ultrasonicator (200 W, 10 minutes) and the disruptedsuspension was centrifuged to obtain granules (inclusion bodies) asprecipitates.

The obtained granules were dissolved in 0.5 M Tris-HCl buffer (pH 8.5)containing 7 M guanidine hydrochloride and 10 mM EDTA. Then, 5 mMreduced type glutathione solution and 0.5 mM oxidized type glutathionesolution were added in a volume of 2.5 times the solution and themixture was left standing overnight at 4° C. The solution was dialyzedagainst 0.9% saline by using a Spectra/Por 1 dialysis membrane (Spectra)to remove guanidine hydrochloride. To the solution after the dialysis,1/9 volume of 0.5 M ammonium acetate buffer (pH 4.5) was added, and themixture was loaded on an ion exchange column using TSK-gel CM-5PW(1.0×7.5 cm) and eluted with Elution solution A (50 mM ammonium acetatebuffer (pH 4.5)) and Elution solution B (0.5 M ammonium acetate buffer(pH 6.4)). The elution was performed with a solution composed of Elutionsolutions A and B (A:B=100:0) for 5 minutes, and then with a lineargradient of from A:B=100:0 to A:B=0:100 for 30 minutes. Thus, purifiedM8-G4 to M20-G4 were obtained. The proteins were quantified by comparingthe elution peak area at 280 nm obtained by reverse phase HPLC usingPegasil ODS-300 (4.6×250 mm, Senshu Kagaku) by using AS1051-Ala (seeExample 1) and AS1051-G4 (see Example 2) quantified beforehand with aBio-Rad Protein Assay (Bio-Rad) as standards.

Example 5

Glycoprotein Ib/von Willebrand Factor Binding Inhibitory Activity ofLoop Structure-deleted Protein (AS1051-G4) and Protein Containing AminoAcid Mutation

The glycoprotein Ib/von Willebrand factor binding inhibitory activity ofthe loop structure-deleted protein (AS1051-G4) prepared in Example 2 andthe proteins having a single amino acid mutation (M7 to M20) prepared inExample 4 was determined in the same manner as in the method of N.Fukuchi et al. (WO99/54360, Example 7 in the specification). That is, 50μl of TBS (Tris-buffered saline, 20 mM Tris-HCl (pH 7.4), 0.15 M NaCl)solution containing von Willebrand factor (2.5 μg/ml) was added to eachwell of a 96-well plate and immobilized overnight at 4° C. as a solidphase. Then, the wells were washed with TBS (150 μl) once and blockedwith TBS containing 5% BSA for 3 hours. The plate was washed with TBS(150 μl) twice. To 25 μl of assay buffer (Assay Buffer, 1244-106,Wallac), a diluted protein whose inhibitory activity was to bedetermined was added. Then, 25 μl of assay buffer containing a chimeraprotein (100 ng/ml) of europium (Eu) labeled human glycoprotein Ibα-chain and a mouse IgG2a Fc region, and botrocetin (500 ng/ml) wereadded thereto and the mixture was left at room temperature for about 3hours. The plate was washed with 150 μl of TBS containing 0.05% Tween-20five times. Then, 100 μl of fluorescence enhancing buffer (Enhancementsolution, 1244-104, Wallac) was added and the plate was shaken for 1minute and then the europium (Eu) amount was measured by using a1420ARVO multi-label counter (Wallac) (measurement time: 1 second). IC₅₀values for glycoprotein Ib/von Willebrand factor binding inhibition ofthe respective mutant proteins are shown in Table 2.

TABLE 2 Protein IC₅₀ value (ng/ml) AS1051-Ala 70.4 AS1051-G4 57.8 M7-G445.4 M8-G4 28.7 M9-G4 1607 M10-G4 701.9 M11-G4 56.0 M12-G4 347.2 M13-G4294.3 M14-G4 1091 M15-G4 234.7 M16-G4 10.6 M17-G4 14327 M18-G4 1292.6M19-G4 26.9 M20-G4 483.0

From the above data, it was found that the inhibitory activity onglycoprotein Ib/von Willebrand factor binding greatly decreased by themutations of Tyr58 (M9-G4), Tyr67 (M14-G4), Arg103 (M17-G4), Arg105(M18-G4) and Phe108 (M20-G4) to Ala and, in particular, the activity wasmost markedly decreased (about 1/250) by the mutation of Arg103(M17-G4). It was also observed that the glycoprotein Ib/von Willebrandfactor binding inhibitory activity was increased by the mutations ofAsp54 (M8-G4), Asp101 (M16-G4) and Glu106 (M19-G4) to Ala. According tothe crystal structure data, the α carbons of all the three residues, ofwhich mutation increased the activity, were within 10 Å from the αcarbon of Arg103, of which mutation most markedly decreased the activityand which was considered to be most important for the glycoproteinIb/von Willebrand factor binding inhibitory activity.

Example 6

Preparation of Highly Active Proteins Containing a Plurality of AminoAcid Mutations and Their Activity

(1) Preparation of Mutant Proteins

Proteins containing a plurality of amino acid mutations were prepared inthe same manner as in the method for preparing the proteins containing asingle amino acid mutation. Designations of mutant genes and theirmutations are shown in Table 3. The amino acid numbers used in the tablecorrespond to the amino acid numbers used in SEQ ID NO: 1.

TABLE 3 Mutant protein Amino acid to be mutated Amino acid aftermutation M21 Asp54, Asp101, Glu106 Ala54, Asn101, Gln106 M22 Asp54,Asp101, Glu106 Ala54, Ala101, Ala106 M23 Asp54, Asp101 Asn54, Ala101 M24Asp54, Asp101 Ala54, Ala101 M25 Asp101, Glu106 Ala101, Gln106 M26Asp101, Glu106 Ala101, Ala106

PCR was performed by using the expression plasmid pTrcM8G4 for M8-G4 asa template, primers M21F (SEQ ID NO: 39) and M21R (SEQ ID NO: 40) orprimers M22F (SEQ ID NO: 41) and M22R (SEQ ID NO: 42) and a QuickChange™Site-Directed Mutagenesis Kit (Stratagene) to obtain expression plasmidspTrcM21G4 and pTrcM22G4 for the mutant protein M21-G4 and M22-G4,respectively. Further, PCR was performed by using the expression plasmidpTrcM16G4 of M16-G4 as a template, primers M23F (SEQ ID NO: 43) and M23R(SEQ ID NO: 44), primers M8F (SEQ ID NO: 13) and M8R (SEQ ID NO: 14),primers M25F (SEQ ID NO: 45) and M25R (SEQ ID NO: 46) or primers M19F(SEQ ID NO: 35) and M19R (SEQ ID NO: 36), and the QuickChange™Site-Directed Mutagenesis Kit (Stratagene) to obtain expression plasmidspTrcM23G4, pTrcM24G4, pTrcM25G4 and pTrcM26G4 for the mutant proteinsM23-G4, M24-G4, M25-G4 and M26-G4, respectively.

The transformant E. coli JM109 strains harboring each of the expressionplasmids pTrcM21G4, pTrcM22G4, pTrcM23G4, pTrcM24G4, pTrcM25G4 andpTrcM26G4 prepared as described above were each cultured at 37° C. inL-broth (1% Bacto trypton, 0.5% yeast extract, 0.5% sodium chloride, 100μg/ml ampicillin sodium) by using Sakaguchi flasks. IPTG(isopropyl-β-thiogalactopyranoside) was added at 10 mM when theturbidity reached 0.5 and further cultured at 37° C. for 4 hours. Themicrobial cells were collected and washed by centrifugation. Then, themicrobial cells were suspended in a 0.5 M EDTA solution. Lysozyme wasadded thereto and the mixture was left standing at room temperature for1 hour. The suspension of the microbial cells was disrupted by anultrasonicator (200 W, 5 minutes) and the disrupted suspension wascentrifuged to obtain granules (inclusion bodies) as precipitates.

The obtained granules were dissolved in 0.5 M Tris-HCI buffer (pH 8.5)containing 7 M guanidine hydrochloride and 10 mM EDTA. Then, distilledwater was added in a volume of 2.5 times the solution and the mixturewas left standing overnight at 4° C. The solution was dialyzed against0.9% saline by using a Spectra/Por 1 dialysis membrane (Spectra) toremove guanidine hydrochloride. To the solution after the dialysis, 1/9volume of 0.5 M ammonium acetate buffer (pH 4.5) was added, and themixture was loaded on an ion exchange column using TSK-gel CM-5PW(1.0×7.5 cm) and eluted with Elution solution A (50 mM ammonium acetatebuffer (pH 4.5)) and Elution solution B (0.5 M ammonium acetate buffer(pH 6.4)). The elution was performed with a solution composed of Elutionsolutions A and B (A:B=100:0) for 5 minutes, and then with a lineargradient of from A:B=100:0 to A:B=0:100 for 30 minutes. Thus, elutedfractions each containing purified M21-G4, M22-G4, M23-G4, M24-G4,M25-G4 or M26G4 were obtained. The protein amount was measured by usingAS1051-Ala (see Example 1) and AS1051-G4 (see Example 2) quantifiedbeforehand by using a Protein Assay Kit (BioRad) as standards andcomparing peak areas at 280 nm obtained in reverse phase HPLC (highperformance liquid chromatography) using Pegasil ODS-300 (4.6×250 mm,Senshu Kagaku).

(2) Measurement of Glycoprotein Ib/von Willebrand Factor BindingInhibitory Activity

Subsequently, the glycoprotein Ib/von Willebrand factor bindinginhibitory activity of the proteins containing a plurality of amino acidmutations was measured by using the method described in Example 5. IC₅₀values of glycoprotein Ib/von Willebrand factor binding inhibition ofthe mutant proteins are shown in Table 4. All the mutant proteinsexhibited increase of the glycoprotein Ib/von Willebrand factor bindinginhibitory activity, and M23-G4 exhibited the highest inhibitoryactivity.

TABLE 4 Protein IC₅₀ value (ng/ml) AS1051-Ala 46.9 AS1051-G4 50.3 M16-G414.2 M21-G4 14.7 M23-G4 9.8 M24-G4 11.6 M25-G4 11.4

Example 7

Preparation of Polyethylene Glycol-modified Proteins and Their Activity

(1) Preparation of Polyethylene-glycolated AS1051 that is not Mutated

Proteins containing amino acid mutations shown in Example 6 to bepolyethylene-glycolated were prepared in the same manner as in theabove-described method.

First, to utilize Cys81 of a natural type AS1051 as Cys to bepolyethylene-glycolated, an expression gene of AS1051 in which Cys81 wasnot replaced with Ala (AS1051-WT) was constructed. PCR was performed byusing the plasmid pCHA1 containing AS1051 described in Example 1 as atemplate and primers ASBN (SEQ ID NO: 4) and ASH (SEQ ID NO: 6). Thereaction product was subjected to agarose gel electrophoresis to extractDNA of 400 base pairs from the gel. This DNA fragment was digested withrestriction enzymes BamHI and HindIII. This DNA fragment was ligatedwith a plasmid pUC18 (Takara Shuzo) digested with restriction enzymesBamHI and HindIII by using a Ligation Kit (Takara Shuzo). The E. coliJM109 strain was transformed with the obtained plasmid by the competentcell method and cultured on an ampicillin-containing plate at 37° C. for16 hours to select a transformant. A plasmid was prepared from a growntransformant by the alkaline SDS method. Construction of the targetplasmid was confirmed by determining the nucleotide sequence by usingthe M13M4 and M13RV primers (both from Takara Shuzo) and a 377PRISM DNAsequencer (Perkin-Elmer). The prepared plasmid was designated aspUCASWT. The plasmid PUCASWT was digested with restriction enzymes NcoIand HindIII and subjected to agarose gel electrophoresis to separate andpurify DNA of 400 base pairs. This DNA fragment was ligated withexpression vector pTrcHisA (Invitrogen) digested with restrictionenzymes NcoI and HindIII by using the Ligation Kit. The E. coli JM109strain was transformed with the obtained plasmid by the competent cellmethod and cultured on an ampicillin-containing plate at 37° C. for 16hours to select a transformant. The expression plasmidcontained in thistransformant was designated as pTrcASWT.

The transformant E. coli JM109 strain harboring the expression plasmidpTrcASWT for AS1051-Cys was cultured in the same manner as in the methodshown in Example 1 to obtain granules (inclusion bodies) asprecipitates.

A polyethylene-glycolated AS1051 protein (AS1051-PEG5000) was preparedby using a polyethylene-glycolating reagent having maleimide groups anda molecular weight of about 5000 (Methoxy-PEG-mal, MW 5000, Item No.:M-MAL-5000, Shearwater Polymers) as follows. The AS1051 granulesobtained by the culture were dissolved in 0.5 M Tris-HCl buffer (pH 8.5)containing 7 M guanidine hydrochloride and 10 mM EDTA. Then, distilledwater was added in a volume 2.5 times the solution and the mixture wasleft standing overnight at 4° C. Further, to this solution, theaforementioned polyethylene-glycolating reagent was added at aconcentration of 0.2 mg/ml and the mixture was left standing at roomtemperature for 3 hours. The solution was dialyzed against distilledwater by using a Spectra/Por 1 dialysis membrane (Spectra) to removeguanidine hydrochloride. To the solution after the dialysis, 1/9 volumeof 0.5 M ammonium acetate buffer (pH 4.5) was added, and the mixture wasloaded on an ion exchange column using CM-TOYOPEARL 650S (2.6×40 cm) andeluted with Elution solution A (50 mM ammonium acetate buffer (pH 4.5))and Elution solution B (0.5 M ammonium acetate buffer (pH 6.4)). Theelution was performed with a linear gradient of from A:B=80:20 toA:B=70:30 (20 minutes) and then with a linear gradient of from A:B=70:30to A:B=55:45 (30 minutes). Thus, an eluted fraction containing purifiedAS1051-PEG5000 was obtained.

A polyethylene-glycolated AS1051 protein (AS1051-PEG20000) was preparedby using a polyethylene-glycolating reagent having a molecular weight ofabout 20,000 (Methoxy-PEG-mal, MW 20000, Item No.: M-MAL-20000,Shearwater Polymers), which had maleimide groups, as follows. The AS1051granules obtained by the culture were dissolved in 0.5 M Tris-HCl buffer(pH 8.5) containing 7 M guanidine hydrochloride and 10 mM EDTA. Then,distilled water was added in a volume 2.5 times the solution and themixture was left standing overnight at 4° C. Further, to this solution,the aforementioned polyethylene-glycolating reagent was added at aconcentration of 0.2 mg/ml and the mixture was left standing at roomtemperature for 3 hours. The solution was dialyzed against distilledwater by using a Spectra/Por 1 dialysis membrane (Spectra) to removeguanidine hydrochloride. To the solution after the dialysis, 1/9 volumeof 0.5 M ammonium acetate buffer (pH 4.5) was added, and the mixture wasloaded on an ion exchange column using CM-TOYOPEARL 650S (2.6×40 cm) andeluted with Elution solution A (50 mM ammonium acetate buffer (pH 4.5))and Elution solution B (0.5 M ammonium acetate buffer (pH 6.4)). Theelution was performed with a linear gradient of from A:B=100:0 toA:B=40:60 (20 minutes) and then with a linear gradient of from A:B=40:60to A:B=0:100 (30 minutes). Thus, an eluted fraction containing purifiedAS1051-PEG20000 was obtained.

(2) Structure of AS1051-PEG5000

SDS electrophoresis revealed that the molecular weight of the obtainedAS1051-PEG5000 was 25 kDa, about 10 kDa larger than that of theAS1051-Ala that was not polyethylene-glycolated (15 kDa). Sincepolyethylene glycol is observed with a size twice as large as theoriginal molecular weight due to hydration in SDS electrophoresis, itwas confirmed that one molecule of polyethylene glycol (molecular weightof about 5000) bonded to one molecule of AS1051-PEG5000. Further, a bandof the AS1051-PEG20000 was observed at about 55 kDa, and it wasconfirmed that one molecule of polyethylene glycol (molecular weight of20,000) bonded to one molecule of AS1051-PEG20000.

Subsequently, the polyethylene glycol-bonding position in AS1051-PEG5000and linkage scheme of disulfide bonds in the other Cys residues weredetermined as follows. AS1051-PEG5000 (100 μg) was digested with lysylendopeptidase (5 μg, Wako Pure Chemical Industries) in 0.1 M Tris-HClbuffer (pH 8.5) containing 2 mM EDTA at 37° C. for 5 hours andfractionated by high performance liquid chromatography using a reversephase column (Vydac 218TP54, Vydac). Elution was performed with a lineargradient of water/acetonitrile containing 0.1% trifluoroacetic acid(TFA) (acetonitrile concentration of from 0 to 50% for 10 minutes,acetonitrile concentration of from 50% to 100% for 5 minutes). Thus,peptide chains obtained by digestion were obtained as Peaks 1 to 6 (FIG.3). The amino acid sequence of each peptide chain was analyzed by usinga protein sequencer Model 476A (Applied Biosystems). Since two Cysresidues were contained in the chain of Peak 3, it was concluded thatthese two Cys residues were coupled to form a disulfide bond. Further,it was concluded that Peak 5 was composed of total three peptide chains,wherein two peptide chains containing one Cys residue and one peptidechain containing two Cys residues were coupled through disulfide bonds.The peptide chains of this Peak 5 were further digested with V8 protease(5 μg, Wako Pure Chemical Industries) in 10 mM ammonium carbonate bufferat 25° C. for 24 hours and fractionated by high performance liquidchromatography using a reverse phase column (Pegasil ODS-II, SenshuKagaku). Elution was performed with a linear gradient ofwater/acetonitrile containing 0.1% TFA (acetonitrile concentration offrom 0 to 50% for 20 minutes). Thus, peptide chains obtained bydigestion were prepared and their amino acid sequences were analyzed. Asa result, it was confirmed that the polyethylene glycol chain was bondedto a Cys residue corresponding to the amino acid number 81 in SEQ ID NO:1 and all the peaks supported that the peptides of Peak 5 had such adisulfide bond as shown in FIG. 3. The bonding scheme of disulfide bondin AS1051-PEG determined as described above was the same as in thereported AS1051 (N. Fukuchi et al., WO95/08573) or other similarproteins originating from snake venom.

(3) Preparation of Polyethylene-glycolated Highly Active Protein

Subsequently, an expression gene for the M23-WT protein containingmutations of Asp54Asn and Asp101Ala as in the case of M23-G4(corresponding to AS1051-WT protein containing two mutations of Asp54Asnand Asp101Ala) was prepared as follows. PCR was performed by using theexpression plasmid pTrcASWT for AS1051 as a template, primers M16F (SEQID NO: 29) and M16R (SEQ ID NO: 30) and a QuickChange™ Site-DirectedMutagenesis Kit (Stratagene) to obtain an expression plasmid pTrcM16WTfor a mutant protein M16-WT. Further, PCR was performed by using theexpression plasmid pTrcM16WT as a template, primers M23F (SEQ ID NO: 43)and M23R (SEQ ID NO: 44) and the QuickChange™ Site-Directed MutagenesisKit (Stratagene) to obtain an expression plasmid pTrcM23WT for themutant protein M23-WT. As in Example 6, the transformant E. coli JM109strain harboring pTrcM23WT was cultured to obtain granules (inclusionbodies) as precipitates. The mutant protein M23-WT obtained as describedabove corresponded to the AS1051-WT protein containing two mutations ofAsp54Asn and Asp101Ala.

Further, an expression gene for a mutant protein M23-Cys, in which oneGly residue closest to the amino terminus among the four Gly residues inM23-G4 was replaced with Cys for polyethylene-glycolation, was preparedas follows. PCR was performed by using the expression plasmid pTrcM23G4for M23-G4 as a template, primers CGGGF (SEQ ID NO: 47) and CGGGR (SEQID NO: 48), and the QuickChange™ Site-Directed Mutagenesis Kit(Stratagene) to obtain an expression plasmid pTrcM23Cys for the mutantprotein M23-Cys. As in Example 6, the transformant E. coli JM109 strainharboring pTrcM23Cys was cultured to obtain granules (inclusion bodies)as precipitates. The mutant protein M23-Cys obtained as described abovecorresponded to a protein in which the Gly residue closest to the aminoterminus in the region of four continuous Gly residues of M23-G4 wasmutated to Cys.

Subsequently, polyethylene-glycolation reaction was performed. First,granules obtained from 2 L of culture broth of the M23-WT were dissolvedin 320 ml of 0.5 M Tris-HCl buffer (pH 8.5) containing 7 M guanidinehydrochloride and 10 mM EDTA. Then, distilled water was added in avolume of 2.5 times the solution and the mixture was left standingovernight at 4° C. Further, to this solution, 22.4 ml of 20 mg/mlaqueous solution of a polyethylene-glycolating reagent having amolecular weight of about 20,000 (Methoxy-PEG-mal, MW 20000, Item No.:M-MAL-20000, Shearwater Polymers) for binding to a thiol group of Cyswas added and the mixture was allowed to react at 4° C. for about 2hours. This solution was dialyzed against distilled water overnight at4° C. by using a Spectra/Por 1 dialysis membrane (Spectra). To theobtained dialyzed solution (1.24 ml), 163.85 g of ammonium sulfate wasadded for salting out. The precipitates were removed by centrifugationand the supernatant was subjected to hydrophobic column chromatographyusing Butyl-Sepharose CL-4B FF (16×150 mm) (Pharmacia). The ammoniumsulfate concentration of the elution solution was lowered from 1 M to 0M (60 minutes) as a linear concentration gradient to collect apolyethylene-glycolated M23 (M23-PEG20000). Further, the collectedfraction was dialyzed against distilled water by using a Spectra/Por 1dialysis membrane (Spectra) to remove ammonium sulfate. To the fractionafter the dialysis, 1/9 volume of 0.5 M ammonium acetate buffer (pH 4.5)was added, and the mixture was loaded on an ion exchange column usingCM-TOYOPEARL 650S (2.6×40 cm) and eluted with Elution solution A (50 mMammonium acetate buffer (pH 4.5)) and Elution solution B (0.5 M ammoniumacetate buffer (pH 6.4)). The elution was performed with a mixedsolution composed of Elution solutions A and B (A:B=80:20) for 20minutes, and then with a linear gradient of from A:B=80:20 to A:B=55:45for 30 minutes. Thus, an eluted fraction containing purifiedM23-PEG20000 was obtained. Further, the eluted fraction was concentratedby ultrafiltration and then purified by gel filtration using SepharoseCL-4B (26×900 mm) to finally obtain about 15 mg of M23-PEG20000. Theprotein concentration was quantified by comparing a peak area at 280 nmobtained in reverse phase HPLC with that of AS1051-Ala having a knownconcentration in the same manner as in Example 6.

Further, M23-PEG5000 bonded with polyethylene glycol having a molecularweight of about 5000 was prepared in the same manner as described aboveby using M-MAL-5000 (Methoxy-PEG-mal, MW5000, Shearwater Polymers)instead of M-MAL-20000. The obtained M23-PEG20000 and M23-PEG5000 showedbands at molecular weights of about 55 kDa and about 25 kDa,respectively, in SDS electrophoresis like AS1051-PEG2000 andAS1051-PEG5000.

Further, polyethylene-glycolation of M23-Cys was similarly performed byusing the obtained granules.

(4) Determination of Glycoprotein Ib/von Willebrand Factor BindingInhibitory Activity of Polyethylene-glycolated Proteins

By using the same method as in Example 5, the glycoprotein Ib/vonWillebrand factor binding inhibitory activity of the preparedpolyethylene-glycolated proteins M23-PEG5000 and M23-PEG20000 werecompared with that of M23-G4 that was not polyethylene-glycolated,AS1051-Ala that was not mutated and polyethylene-glycolated AS1051. Asshown in FIG. 4 and Table 5, all the proteins obtained by mutation ofM23 exhibited the inhibitory activity about 10 times as strong as thatof the others, and further increase of the inhibitory activity wasobtained by polyethylene-glycolation.

TABLE 5 Protein IC₅₀ value (ng/ml) AS1051-Ala 54.4 AS1051-PEG5000 98.1AS1051-PEG20000 90.3 M23-G4 7.39 M23-PEG5000 5.96 M23-PEG20000 5.55

Further, the inhibitory activity of the above proteins for humanplatelet aggregation induced by ristocetin, ADP (adenosine diphosphate),collagen and low-concentration thrombin was determined. The humanplatelet rich plasma (PRP) was prepared as follows. Blood was collectedfrom healthy volunteers by using an 18G injection needle. To thecollected blood, 3.8% sodium citrate was added in 1/10 volume of theblood, and the mixture was centrifuged by a centrifugal machine underconditions of 900 rpm, 15 minutes and room temperature to collect thesupernatant as PRP. The lower layer was further centrifuged underconditions of 1500 rpm, 10 minutes and room temperature to collect thesupernatant as platelet poor plasma (PPP). Then, the plateletaggregation inhibitory activity of the above proteins was determined byusing PRP prepared as described above and a Hematracer 801 (NikoBioscience) as a measurement apparatus. To a special cuvette containingabout 2.5 μl of a 20-fold solution of a protein to be determined, 100 μlof PRP was added, and the cuvette was set on the measurement apparatus,shaken for 2 minutes (37° C.). Then, a solution of anaggregation-inducing substance at 10-fold concentration, and changes intransmitted light were measured. The aggregation ratio was calculated onthe assumption that the transmittance of read light before addition ofthe aggregation inducing substance was 0% and that the transmittance ofPPP was 100%

As a result of the measurement, all the proteins exhibited nosubstantial inhibitory activity for aggregation induced by ADP andcollagen, but a strong inhibitory activity for aggregation induced byristocetin. The mutated M23 proteins exhibited stronger inhibitoryactivity. The aggregation curve of ristocetin-induced aggregation isshown in FIG. 5.

Further, the inhibitory activity for aggregation induced by lowconcentration thrombin was determined as follows. To 5 ml of PRPprepared in the same manner as described above, apyrase (grade VII,sigma) was added at a concentration of 8.3 U/ml and the mixture wasincubated at 37° C. for 15 minutes. The reaction mixture was centrifugedat 2000 rpm for 10 minutes, and precipitated platelets were carefullysuspended in a Tyrode-HEPES buffer containing apyrase (4.2 U/ml) andfurther centrifuged at 200 rpm for 10 minutes. Finally, precipitatedplatelets were suspended in a Tyrode-HEPES buffer containing 5 ml of 2mM calcium chloride to obtain a washed platelet solution. Aggregationinhibition was measured by using the washed platelet solution instead ofPRP and Tyrode-HEPES buffer instead of PPP by the same method asdescribed above. The final concentration of thrombin was 0.07 U/ml. As aresult, AS1051-Ala exhibited no substantial inhibition at aconcentration of 4 μg/ml, whereas M23-PEG20000 almost completelyinhibited thrombin-induced aggregation at a concentration of 1 μg/ml orhigher (FIG. 6).

Example 8

Preparation of M23-Ala (Mutant M23 Protein that is not Shortened Protein(G4))

Further, a protein corresponding to M23-WT in which the Cys in position81 in the amino acid sequence is mutated to Ala, M23-Ala, was preparedas follows. PCR was performed by using the expression plasmid pTrcM23WTfor M23-WT as a template, primers ALAF-2 (SEQ ID NO: 49) and ALAR-2 (SEQID NO: 50) and a QuickChange™ Site-Directed Mutagenesis Kit (Stratagene)to obtain an expression plasmid pTrcM23Ala for M23-Ala. The transformantE. coli JM109 strain harboring the prepared expression plasmidpTrcM23Ala was cultured in L-broth (1% Bacto trypton, 0.5% yeastextract, 0.5% sodium chloride, 100 μg/ml ampicillin sodium) at 37° C. byusing a Sakaguchi flask. IPTG (isopropyl-β-thiogalactopyranoside) wasadded at 10 mM when the turbidity reached 0.5 and further cultured at37° C. for 4 hours. The microbial cells were collected and washed bycentrifugation. Then, the microbial cells were suspended in 0.5 M EDTAsolution. Lysozyme was added thereto and then left standing at roomtemperature for 1 hour. The suspension of the microbial cells wasdisrupted by an ultrasonicator (200 W, 5 minutes), and the disruptedsuspension was centrifuged to obtain granules (inclusion bodies) asprecipitates.

The obtained granules were dissolved in 0.5 M Tris-HCl buffer (pH 8.5)containing 7 M guanidine hydrochloride and 10 mM EDTA. Then, distilledwater was added in a volume 2.5 times the solution and the mixture wasleft standing overnight at 4° C. The solution was dialyzed against 0.9%saline by using a Spectra/Por 1 dialysis membrane (Spectra) to removeguanidine hydrochloride. To the solution after the dialysis, 1/9 volumeof 0.5 M ammonium acetate buffer (pH 4.5) was added, and the mixture wasloaded on an ion exchange column using CM-TOYOPEARL 650S (2.6×40 cm) andeluted with Elution solution A (50 mM ammonium acetate buffer (pH 4.5))and Elution solution B (0.5 M ammonium acetate buffer (pH 6.4)). Theelution was performed with a solution composed of Elution solutions Aand B (A:B=30:70) for 20 minutes, and then with a linear gradient offrom A:B=30:70 to A:B=0:100 for 30 minutes. Thus, an eluted fractioncontaining purified M23-Ala was obtained. The protein was quantified bycomparing elution peak areas at 280 nm obtained by reverse phase HPLCusing Pegasil ODS-300 (4.6×250 mm, Senshu Kagaku) by using AS1051-Ala(see Example 1) and AS1051-G4 (see Example 2) quantified beforehandusing a Bio-Rad Protein Assay (Bio-Rad) as standards. When theglycoprotein Ib/von Willebrand factor binding inhibitory activity ofM23-Ala was determined in the same manner as in Examples 5 and 6, theinhibitory activity was almost comparable to that of M23.

Example 9

Antigenicity Test of AS1051-Ala in Guinea Pig

To Confirm antigenicity in a guinea pig of the AS1051 protein in whichcysteine residue in position 81 was replaced with an Ala residue(AS1051-Ala), which was prepared as shown in Example 1, a test wasperformed as follows. Two groups of female Hartley guinea pigs (bodyweight 200 to 250 g) were administered with AS1051-Ala (300 μg/kg) orphysiological saline from auricular veins three times every other day.The dose was 1 ml/kg and each group was composed of 10 animals (n=10).Following 3 weeks after the third administration, each administrationgroup was further divided into two groups, each of which wasadministered with AS1051-Ala (300 μg/kg) (n=5 each) or physiologicalsaline (n=5 each). About 20 minutes later, abdominal section wasperformed under etherization and 8 ml of blood was collected from theabdominal aorta (0.38% sodium citrate was added) by using a 23Ginjection needle. The number of platelets in the collected blood wasmeasured by using an automatic cell counter (Sysmex E-2000, Toa MedicalElectronics). The results are shown in FIG. 7. A marked decrease ofplatelets was observed only in the group in which AS1051-Ala wasadministered at preliminary and final administrations (AS/AS group). Inthe group in which physiological saline was administered at preliminaryadministration and AS1051-Ala was administered at final administration(saline/AS group), the number of platelets was substantially the same asthat of the saline/saline group as a control group. Therefore, it wasconsidered that the decrease of platelets observed in the AS/AS groupwas attributable to the antigenicity of AS1051-Ala administered atpreliminary administration.

Further, plasma was separated from the collected blood by centrifugation(4° C., 2700 rpm, 10 minutes), and the presence of antibodies toAS1051-Ala was determined by the enzyme-linked immunosorbent assay(ELISA) method. 50 μl of AS1051-Ala (1 μg/ml) or only a buffer was addedto each well of a 96-well plate for ELISA and left standing overnight at4° C. to coat the well. Then, each well was washed three times withphosphate-buffered saline (PBS) containing 0.05% Tween-20 and blockedwith PBS (150 μl) dissolving 5% skim milk. Each well was further washedthree times. Then, 50 μl of the collected guinea pig plasma was addedthereto and the plate was left at 37° C. for 1 hour. Then, each well waswashed three times. 50 μl of a solution obtained by diluting alkalinephosphatase-labeled rabbit anti-guinea pig IgG (H+L) antibodies (Zymed)500-fold with a dilution buffer (0.05 M Tris-HCl (pH 8.1), 1 mM MgCl₂,0.15 M NaCl, 0.05% Tween-20, 0.02% NaN₃, 1% bovine serum albumin) wasadded thereto and the plate was left at 37° C. for 1 hour. Each well waswashed three times and 1 mg/ml of chromogenic substrate(p-nitrophenylphosphate) solution (1 M diethanolamine (pH 9.8)/0.5 mMMgCl₂) was added. After an appropriate time for the reaction, absorptionwas measured at 405 nm. FIG. 8 shows absorption of reaction mixtures inthe wells coated with AS1051-Ala and the wells without coating for theAS/saline group and the saline/saline group. As shown in the figure, thepresence of antibodies bound to AS1051-Ala was demonstrated in theAS/saline group.

Example 10

Antigenicity Test of AS1051-G4 in Guinea Pig

A test in a guinea pig for comparing antigenicity of the loopstructure-deleted protein (AS1051-G4) and polyethylene-glycolatedprotein (AS1051-PEG5000) according to the present invention andAS1051-Ala was performed in the same manner as in the method describedin Example 9. The doses of AS1051-Ala and AS1051-G4 were 200 μg/kg forany administration. The subjects were divided into four groups of theAS1051-Ala group (three times of preliminary administration ofAS1051-Ala and final administration of AS1051-Ala), AS1051-G4 group(three times of preliminary administration of AS1051-G4 and finaladministration of AS1051-G4), AS1051-PEG5000 group (three times ofpreliminary administration of AS1051-PEG5000 and final administration ofAS1051-PEG5000) and saline group (three times of preliminaryadministration of physiological saline and final administration ofphysiological saline), each being composed of 4 animals, to perform anexperiment. As a result, as shown in FIG. 9, both of the AS1051-G4 groupand the AS1051-PEG5000 group exhibited weaker platelet decrease actioncompared with that of the AS1051-Ala group.

Example 11

Ex vivo Drug Efficacy Test of Polyethylene-glycolated Highly ActiveProtein

An ex vivo drug efficacy test of M23-PEG20000 in guinea pig wasperformed as follows. First, M23-PEG20000 physiological saline solutionswere prepared at various concentrations, and female Hartley guinea pigs(body weight 350 to 400 g) were administered with 1 ml/kg ofM23-PEG20000 solution at each concentration or physiological saline as acontrol group from auricular veins. Five minutes or 5 days (120 hours)after the administration, abdominal section was performed underetherization and blood was collected from the abdominal aorta (0.38%sodium citrate was added) by using a 23G injection needle. The number ofplatelets in the collected blood was measured by using an automatic cellcounter (Sysmex E-2000, Toa Medical Electronics). Further, the collectedblood was centrifuged under conditions of 1100 rpm, 15 minutes and roomtemperature to prepare PRP. Further, the lower layer was centrifugedunder conditions of 2700 rpm, 15 minutes and room temperature to preparePPP. To the obtained PRP, botrocetin was added as a platelet aggregationinducing substance, and the maximum botrocetin-induced aggregation ratein 8 minutes was measured for each group by using the same plateletaggregometer as used in the method of Example 7. As a result, among thegroups in which blood was collected 5 minutes after the administrationof M23-PEG20000, the 100 μg/kg administered group exhibited asignificant inhibitory effect on aggregation as shown in FIG. 10(a).Further, the 1 mg/kg administered group exhibited strong inhibition onbotrocetin-induced aggregation even in the blood collected 5 days (120hours) after the administration (FIG. 10(b)). Thus, it was confirmedthat the inhibitory effect on glycoprotein Ib/von Willebrand factorbinding was maintained even for 5 days (120 hours) after theadministration at that dose. Further, no significant change in thenumber of blood cells was observed in all of the groups.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 50 <210> SEQ ID NO 1 <211> LENGTH: 126<212> TYPE: PRT <213> ORGANISM: Crotallus horridus <400> SEQUENCE: 1Asp Leu Glu Cys Pro Ser Gly Trp Ser Ser Th #r Asp Arg Tyr Cys Tyr1               5    #                10   #                15Lys Pro Phe Lys Gln Glu Met Thr Trp Ala Se #r Ala Glu Arg Phe Cys            20       #            25       #            30Ser Glu Gln Ala Lys Gly Gly His Leu Leu Se #r Val Glu Thr Ala Leu        35           #        40           #        45Glu Ala Ser Phe Val Asp Asn Val Leu Tyr Al #a Asn Lys Glu Tyr Leu    50               #    55               #    60Thr Arg Tyr Ile Trp Ile Gly Leu Arg Val Gl #n Asn Lys Gly Gln Pro65                   #70                   #75                   #80Cys Ser Ser Ile Ser Tyr Glu Asn Leu Val As #p Pro Phe Glu Cys Phe                85   #                90   #                95Met Val Ser Arg Asp Thr Arg Leu Arg Glu Tr #p Phe Lys Val Asp Cys            100       #           105       #           110Glu Gln Gln His Ser Phe Ile Cys Lys Phe Th #r Arg Pro Arg        115           #       120           #       125<210> SEQ ID NO 2 <211> LENGTH: 690 <212> TYPE: DNA<213> ORGANISM: Crotalus harridus <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (66)..(512) <223> OTHER INFORMATION: <400> SEQUENCE: 2ctgagcagac ttgctacctg tggaggccga ggaacagttc tctctgcagg ga#aggaaaga     60 acgcc atg ggg cga ttc atc ttc gtg agc ttc #aac ttg ctg gtc gtg ttc    110       Met Gly Arg Phe Ile Phe Val #Ser Phe Asn Leu Leu Val Val Phe       1            #   5                #    10               #    15ctc tcc cta agt gga act cta gct gat ttg ga#a tgt ccc tcc ggt tgg      158Leu Ser Leu Ser Gly Thr Leu Ala Asp Leu Gl #u Cys Pro Ser Gly Trp                20   #                25   #                30tct tcc tat gat cgg tat tgc tac aag ccc tt#c aaa caa gag atg acc      206Ser Ser Tyr Asp Arg Tyr Cys Tyr Lys Pro Ph #e Lys Gln Glu Met Thr            35       #            40       #            45tgg gcc gat gca gag agg ttc tgc tcg gag ca#g gcg aag ggc ggg cat      254Trp Ala Asp Ala Glu Arg Phe Cys Ser Glu Gl #n Ala Lys Gly Gly His        50           #        55           #        60ctc ctc tct gtc gaa acc gcc cta gaa gca tc#c ttt gtg gac aat gtg      302Leu Leu Ser Val Glu Thr Ala Leu Glu Ala Se #r Phe Val Asp Asn Val    65               #    70               #    75ctc tat gcg aac aaa gag tac ctc aca cgt ta#t atc tgg att gga ctg      350Leu Tyr Ala Asn Lys Glu Tyr Leu Thr Arg Ty #r Ile Trp Ile Gly Leu80                   #85                   #90                   #95agg gtt caa aac aaa gga cag cca tgc tcc ag#c atc agt tat gag aac      398Arg Val Gln Asn Lys Gly Gln Pro Cys Ser Se #r Ile Ser Tyr Glu Asn                100   #               105   #               110ctg gtt gac cca ttt gaa tgt ttt atg gtg ag#c aga gac aca agg ctt      446Leu Val Asp Pro Phe Glu Cys Phe Met Val Se #r Arg Asp Thr Arg Leu            115       #           120       #           125cgt gag tgg ttt aaa gtt gac tgt gaa caa ca#a cat tct ttc ata tgc      494Arg Glu Trp Phe Lys Val Asp Cys Glu Gln Gl #n His Ser Phe Ile Cys        130           #       135           #       140aag ttc acg cga cca cgt taagatccgg ctgtgtgaag tc#tggagaag             542 Lys Phe Thr Arg Pro Arg     145caaggaagcc ccccacctct ccccaccccc caccttccgc aatctctgct ct#tccccctt    602tgctcagtgg atgctctctg tagccggatc tgggttttct gctccagatg gg#tcagaaga    662 tccaataaat tctgcctacc caaaaaaa         #                   #            690 <210> SEQ ID NO 3 <211> LENGTH: 149<212> TYPE: PRT <213> ORGANISM: Crotalus harridus <400> SEQUENCE: 3Met Gly Arg Phe Ile Phe Val Ser Phe Asn Le #u Leu Val Val Phe Leu1               5    #                10   #                15Ser Leu Ser Gly Thr Leu Ala Asp Leu Glu Cy #s Pro Ser Gly Trp Ser            20       #            25       #            30Ser Tyr Asp Arg Tyr Cys Tyr Lys Pro Phe Ly #s Gln Glu Met Thr Trp        35           #        40           #        45Ala Asp Ala Glu Arg Phe Cys Ser Glu Gln Al #a Lys Gly Gly His Leu    50               #    55               #    60Leu Ser Val Glu Thr Ala Leu Glu Ala Ser Ph #e Val Asp Asn Val Leu65                   #70                   #75                   #80Tyr Ala Asn Lys Glu Tyr Leu Thr Arg Tyr Il #e Trp Ile Gly Leu Arg                85   #                90   #                95Val Gln Asn Lys Gly Gln Pro Cys Ser Ser Il #e Ser Tyr Glu Asn Leu            100       #           105       #           110Val Asp Pro Phe Glu Cys Phe Met Val Ser Ar #g Asp Thr Arg Leu Arg        115           #       120           #       125Glu Trp Phe Lys Val Asp Cys Glu Gln Gln Hi #s Ser Phe Ile Cys Lys    130               #   135               #   140 Phe Thr Arg Pro Arg145 <210> SEQ ID NO 4 <211> LENGTH: 30 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 4attggatcca tggatttgga atgtccctcc          #                  #           30 <210> SEQ ID NO 5 <211> LENGTH: 26 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 5ggacagccag cctccagcat cagtta           #                  #              26 <210> SEQ ID NO 6 <211> LENGTH: 30 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 6aataagctta acgtggtcgc gtgaacttgc          #                  #           30 <210> SEQ ID NO 7 <211> LENGTH: 26 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 7gatgctggag gctggctgtc ctttgt           #                  #              26 <210> SEQ ID NO 8 <211> LENGTH: 54 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 8tatatctgga ttggactgag gggcggtgga ggtgaatgtt ttatggtgag ca#ga           54 <210> SEQ ID NO 9 <211> LENGTH: 54 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 9tctgctcacc ataaaacatt cacctccacc gcccctcagt ccaatccaga ta#ta           54 <210> SEQ ID NO 10 <211> LENGTH: 110 <212> TYPE: PRT<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC PEPTIDE <400> SEQUENCE: 10Asp Leu Glu Cys Pro Ser Gly Trp Ser Ser Ty #r Ser Arg Tyr Cys Tyr1               5    #                10   #                15Lys Pro Phe Lys Gln Glu Met Thr Tyr Ala As #p Ala Glu Arg Phe Cys            20       #            25       #            30Ser Glu Gln Ala Lys Gly Gly His Leu Leu Se #r Val Glu Thr Ala Leu        35           #        40           #        45Glu Ala Ser Phe Val Asp Asn Val Leu Tyr Al #a Asn Lys Glu Tyr Leu    50               #    55               #    60Thr Arg Tyr Ile Trp Ile Gly Leu Arg Phe Ph #e Phe Phe Glu Cys Phe65                   #70                   #75                   #80Met Val Ser Arg Asp Thr Arg Leu Arg Glu Tr #p Phe Lys Val Asp Cys                85   #                90   #                95Glu Gln Gln His Ser Phe Ile Cys Lys Phe Th #r Arg Pro Arg            100       #           105       #           110<210> SEQ ID NO 11 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 11caagcccttc gcacaagaga tgac           #                  #                24 <210> SEQ ID NO 12 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 12gtcatctctt gtgcgaaggg cttg           #                  #                24 <210> SEQ ID NO 13 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 13gcatcctttg tggccaagtg gctc           #                  #                24 <210> SEQ ID NO 14 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 14gagcacattg gccacaaagg atgc           #                  #                24 <210> SEQ ID NO 15 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 15gacaatgtgc tcgctgcgaa caaag           #                  #               25 <210> SEQ ID NO 16 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 16ctttgttcgc agcgagcaca ttgtc           #                  #               25 <210> SEQ ID NO 17 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 17ctatgcgaac gcagagtacc tcac           #                  #                24 <210> SEQ ID NO 18 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 18gtgaggtact ctgcgttcgc atag           #                  #                24 <210> SEQ ID NO 19 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 19gcgaacaaag cgtacctcac acg            #                  #                23 <210> SEQ ID NO 20 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 20cgtgtgaggt acgctttgtt cgc            #                  #                23 <210> SEQ ID NO 21 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 21gcgaacaaag aggccctcac acgt           #                  #                24 <210> SEQ ID NO 22 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 22acgtgtgagg gcctctttgt tcgc           #                  #                24 <210> SEQ ID NO 23 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 23gtacctcaca gcttatatct gg            #                  #                 22 <210> SEQ ID NO 24 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 24ccagatataa gctgtgaggt ac            #                  #                 22 <210> SEQ ID NO 25 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 25cctcacacgt gctatctgga ttgg           #                  #                24 <210> SEQ ID NO 26 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 26ccaatccaga tagcacgtgt gagg           #                  #                24 <210> SEQ ID NO 27 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 27atggtgagcg cagacacaag gc            #                  #                 22 <210> SEQ ID NO 28 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 28gccttgtgtc tgcgctcacc at            #                  #                 22 <210> SEQ ID NO 29 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 29ggtgagcaga gccacaaggc ttcg           #                  #                24 <210> SEQ ID NO 30 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 30cgaagccttg tggctctgct cacc           #                  #                24 <210> SEQ ID NO 31 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 31agagacacag cgcttcgtga ggc            #                  #                23 <210> SEQ ID NO 32 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 32ctcacgaagc gctgtgtctc tgc            #                  #                23 <210> SEQ ID NO 33 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 33gaacaaggct tgctgagtgg tttaaag           #                  #             27 <210> SEQ ID NO 34 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 34ctttaaacca ctcagcaagc cttgttc           #                  #             27 <210> SEQ ID NO 35 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 35caaggcttcg tgcgtggttt aaagttg           #                  #             27 <210> SEQ ID NO 36 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 36caactttaaa ccacgcacga agccttg           #                  #             27 <210> SEQ ID NO 37 <211> LENGTH: 26 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 37cttcgtgagt gggctaaagt tgactg           #                  #              26 <210> SEQ ID NO 38 <211> LENGTH: 26 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 38cagtcaactt tagcccactc acgaag           #                  #              26 <210> SEQ ID NO 39 <211> LENGTH: 41 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 39ggtgagcaga aacacaaggc ttcgtcagtg gtttaaagtt g     #                  #   41 <210> SEQ ID NO 40 <211> LENGTH: 41 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 40caactttaaa ccactgacga agccttgtgt ttctgctcac c     #                  #   41 <210> SEQ ID NO 41 <211> LENGTH: 41 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 41ggtgagcaga gccacaaggc ttcgtgcgtg gtttaaagtt g     #                  #   41 <210> SEQ ID NO 42 <211> LENGTH: 41 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 42caactttaaa ccacgcacga agccttgtgg ctctgctcac c     #                  #   41 <210> SEQ ID NO 43 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 43gcatcctttg tgaacaatgt gctc           #                  #                24 <210> SEQ ID NO 44 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 44gagcacattg ttcacaaagg atgc           #                  #                24 <210> SEQ ID NO 45 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 45caaggattcg tcagtggttt aaagttg           #                  #             27 <210> SEQ ID NO 46 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 46caactttaaa ccactgacga agccttg           #                  #             27 <210> SEQ ID NO 47 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 47ggattggact gaggtgcggt ggagg           #                  #               25 <210> SEQ ID NO 48 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 48cctccaccgc acctcagtcc aatcc           #                  #               25 <210> SEQ ID NO 49 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 49ggacagccag catccagcat cag            #                  #                23 <210> SEQ ID NO 50 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:<223> OTHER INFORMATION: SYNTHETIC DNA <400> SEQUENCE: 50ctgatgctgg atgctggctg tcc            #                  #                23

What is claimed is:
 1. A protein comprising an amino acid sequence witha homology of not less than 30% to the amino acid sequence of SEQ ID NO:1 and forms a secondary or tertiary structure composed of a first βstrand (β1), a first α helix (α1), a second α helix (α2), a second βstrand (β2), a loop, a third β strand (β3), a fourth β strand (β4) and afifth β strand (β5) in this order from the amino terminus, and whereinat least one amino acid residue in a region from α2 to β2, a region fromβ3 to β4, or in the regions from α2 to β2 and from β3 to β4 is replacedso that electric charge of the amino acid residue is changed towardspositive direction as compared to the unsubstituted amino acid, saidprotein being the following (a) or (b): (a) a protein, in which theregion from α2 to β2 has the sequence of the amino acid residues 47 to72 in the amino acid sequence of SEQ ID NO: 1 and the region from β3 toβ4 has the sequence of the amino acid residues 94 to 111 in the aminoacid sequence of SEQ ID NO: 1; (b) the protein according to (a), inwhich substitution, insertion or deletion of one amino acid residue isincluded in the region from α2 to β2 having the sequence of the aminoacid residues 47 to 72 in the amino acid sequence of SEQ ID NO: 1, orthe region from β3 to β4 having the sequence of the amino acid residues94 to 111 in the amino acid sequence of SEQ ID NO: 1; and wherein saidprotein has antithrombotic activity.
 2. The protein according to claim1, wherein the protein is bonded to a polyoxyalkylpolyol group via areactive functional group wherein said reactive functional group isselected from the group consisting of an amino reactive functionalgroup, a carboxyl reactive functional group, and a thiol reactivefunctional group.
 3. The protein according to claim 1, wherein theprotein is bonded to a polyoxyalkylpolyol group via a thiol reactivefunctional group.
 4. The protein according to claim 3, wherein saidthiol reactive functional group is selected from the group consisting ofa maleimide group, an orthopyridyl disulfide group, and a vinylsulfonegroup.
 5. A protein comprising an amino acid sequence with a homology ofnot less than 30% to the amino acid sequence of SEQ ID NO: 1 and forms asecondary or tertiary structure composed of a first β strand (β1), afirst α helix (α1), a second α helix (α2), a second β strand (β2), aloop, a third β strand (β3), a fourth β strand (β4) and a fifth β strand(β5) in this order from the amino terminus, and wherein at least oneamino acid residue in a region from α2 to β2, a region from β3 to β4, orin the regions from α2 to β2 and from β3 to β4 is substituted so thatelectric charge of the amino acid residue is changed towards positivedirection as compared to the unsubstituted amino acid, said proteinbeing the following (a) or (b): (a) a protein, in which the region fromα2 to β2 has the sequence of the amino acid residues 47 to 72 in theamino acid sequence of SEQ ID NO: 1 and the region from β3 to β4 has thesequence of the amino acid residues 94 to 111 in the amino acid sequenceof SEQ ID NO: 1; (b) the protein according to (a), in whichsubstitution, insertion or deletion of one amino acid residue isincluded in the region from α2 to β2 having the sequence of the aminoacid residues 47 to 72 in the amino acid sequence of SEQ ID NO: 1, orthe region from β3 to β4 having the sequence of the amino acid residues94 to 111 in the amino acid sequence of SEQ ID NO: 1; and wherein saidprotein has antithrombotic activity, and wherein said protein comprisesan amino acid sequence of the following (A) or (B): (A) the amino acidsequence of the amino acid residues 47 to 111 in the amino acid sequenceof SEQ ID NO: 1; (B) the amino acid sequence according to (A), in whichthe cysteine residue of the amino acid residue 81 in the amino acidsequence of SEQ ID NO: 1 is replaced with an alanine residue.
 6. Aprotein comprising an amino acid sequence with a homology of not lessthan 30% to the amino acid sequence of SEQ ID NO: 1 and forms asecondary or tertiary structure composed of a first β strand (β1), afirst α helix (α1), a second α helix (α2), a second β strand (β2), aloop, a third β strand (β3), a fourth β strand (β4) and a fifth β strand(β5) in this order from the amino terminus, and wherein at least oneamino acid residue in a region from α2 to β2, a region from β3 to β4, orin the regions from α2 to β2 and from β3 to β4 is substituted so thatelectric charge of the amino acid residue is changed towards positivedirection as compared to the unsubstituted amino acid, said proteinbeing the following (a) or (b): (a) a protein, in which the region fromα2 to β2 has the sequence of the amino acid residues 47 to 72 in theamino acid sequence of SEQ ID NO: 1 and the region from β3 to β4 has thesequence of the amino acid residues 94 to 111 in the amino acid sequenceof SEQ ID NO: 1; (b) the protein according to (a), in whichsubstitution, insertion or deletion of one amino acid residue isincluded in the region from α2 to β2 having the sequence of the aminoacid residues 47 to 72 in the amino acid sequence of SEQ ID NO: 1, orthe region from β3 to β4 having the sequence of the amino acid residues94 to 111 in the amino acid sequence of SEQ ID NO: 1; and wherein saidprotein has antithrombotic activity, and wherein said protein has theamino acid sequence in which a region containing the loop structureexisting between β2 and β3 is deleted in such a manner that thesecondary or tertiary structures of β2 and β3 are maintained, or theregion is replaced with one or more amino acid residue(s) in a numberrequired to maintain the secondary or tertiary structures of β2 and β3,said amino acid residue(s) being selected from the group consisting of aglycine residue, an alanine residue, a serine residue and a cysteineresidue.
 7. The protein according to claim 6, wherein the regioncontaining the loop structure existing between β2 and β3 is replacedwith an amino acid sequence composed of four glycine residues.
 8. Aprotein comprising an amino acid sequence with a homology of not lessthan 30% to the amino acid sequence of SEQ ID NO: 1 and forms asecondary or tertiary structure composed of a first β strand (β1), afirst α helix (α1), a second α helix (α2), a second β strand (β2), aloop, a third β strand (β3), a fourth β strand (β4) and a fifth β strand(β5) in this order from the amino terminus, and wherein at least oneamino acid residue in a region from α2 to β2, a region from β3 to β4, orin the regions from α2 to β2 and from β3 to β4 is substituted so thatelectric charge of the amino acid residue is changed towards positivedirection as compared to the unsubstituted amino acid, said proteinbeing the following (a) or (b): (a) a protein, in which the region fromα2 to β2 has the sequence of the amino acid residues 47 to 72 in theamino acid sequence of SEQ ID NO: 1 and the region from β3 to β4 has thesequence of the amino acid residues 94 to 111 in the amino acid sequenceof SEQ ID NO: 1; (b) the protein according to (a), in whichsubstitution, insertion or deletion of one amino acid residue isincluded in the region from α2 to β2 having the sequence of the aminoacid residues 47 to 72 in the amino acid sequence of SEQ ID NO: 1, theregion from β3 to β4 having the sequence of the amino acid residues 94to 111 in the amino acid sequence of SEQ ID NO: 1 and wherein saidprotein has antithrombotic activity, and wherein at least one acidicamino acid residue of which α carbon atom exists within 10 Å from the αcarbon atom of the arginine residue of the amino acid number 103 in theamino acid sequence of SEQ ID NO: 1 is replaced with a neutral aminoacid residue.
 9. The protein according to claim 8, wherein the acidicamino acid residue to be replaced is composed of at least one residueselected from the aspartic acid residue of the amino acid residue 54,the aspartic acid of the amino acid residue 101 and the glutamic acidresidue of the amino acid residue 106 in the amino acid sequence of SEQID NO:
 1. 10. The protein according to claim 1, wherein the protein isbonded to a polyoxyalkylpolyol group.
 11. The protein according to claim10, wherein the protein contains a cysteine residue corresponding to acysteine residue of the amino acid residue 81 in the amino acid sequenceof SEQ ID NO: 1 and the polyoxyalkylpolyol group is bonded to saidcysteine residue.
 12. The protein according to claim 10, wherein thepolyoxyalkylpolyol group is a polyethylene glycol group.
 13. A drugcomprising the protein as claimed in claim 1 as an active ingredient.14. A drug comprising the protein as claimed in claim 5 as an activeingredient.
 15. A drug comprising the protein as claimed in claim 6 asan active ingredient.
 16. A drug comprising the protein as claimed inclaim 7 as an active ingredient.
 17. A drug comprising the protein asclaimed in claim 8 as an active ingredient.
 18. A drug comprising theprotein as claimed in claim 9 as an active ingredient.
 19. A drugcomprising the protein as claimed in claim 10 as an active ingredient.